Granulomatous Infections: Etiology and Classification...Granulomatous Infections: Etiology and...

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Granulomatous Infections: Etiology and Classification

Alimuddin Zumla and D. Geraint James From the Academic Infectious Diseases Unit, Department ofMedicine,University College London Medical School; and the Royal Free

Hospital School ofMedicine, London, United Kingdom

Granulomatous disorders are frequently due to a wide variety of infections. Over the past decadeadvances in molecular diagnostic techniques have allowed identification of organisms involved ingranulomatous disorders that previously were of unknown etiol~gy. ~n the basis of cu~ently available information, granulomatous infections can now be classified m three categones. Group 1infections are due to a well-recognized organism. Group 2 comprises infections due to organismsthat have been recently identified in granulomas by molecular methods but are not readily isolatedby conventional microbiological techniques. Group 3 consists of disorders for which the causalorganisms have not yet been identified but are strongly suspected; further a~van~es in di~gnostic

techniques will lead to reclassification of some of these disorders as grou~ 2. ThIs r~vlew descnbe~ theetiology,histopathologic features, and classificationof granulomatous disorders, with an emphasis onthose of groups 2 and 3.

Granuloma Formation

A granuloma can be defined as a focal, compact collectionof inflammatory cells in which mononuclear cells predominate.Granulomas usually form as a result of the persistence of anondegradable product or as the result of hypersensitivity responses [1]. An overlap of the two mechanisms occurs in mostinfectious diseases since microorganisms can serve both asforeign bodies and as antigens for immunologic responses.Normally granulomas are the result of protective mechanismsand form when acute inflammatory processes cannot destroyinvading agents.

The granuloma forms by a stepwise series of events and isthe end result ofa complex interplay among invading organism,antigen, chemical, drug or other irritant, prolonged antigenemia, macrophage activity, T cell responses, B cell overactivity,circulating immune complexes, and a vast array of biologicalmediators [2]. Areas ofinflammation or immunologic reactivityattract monocyte-macrophages, which may fuse to form multinucleated giant cells [3]. Further cellular transformation ofmacrophages to epithelioid cells may occur.

The granuloma is an active site of numerous enzymes andcytokines and, with aging, fibronectin and progression factorssuch as platelet-derived growth factor, transforming growthfactor-,B, insulin-like growth factor, and TNF-a. There is aclose relationship between CD4+ T lymphocytes and activatedmacrophages showing increased expression ofmajor histocom-

Received 21 August 1995; revised 23 February 1996.Financial support: Camden and Islington Community Health Services NHS

Trust.Reprints or correspondence: Professor D. Geraint James, Visiting ~rofessor

of Medicine, Royal Free Hospital School of Medicine, Rowland HIll Street,London NW3 2PF, United Kingdom

Clinical Infectious Diseases 1996;23:146-58© 1996 by The University of Chicago. All rights reserved.1058-4838/96/2301-0020$02.00

patibility (MHC) class II molecules. The T helper cells recognize protein peptides presented to it by antigen-presenting cellsbearing MHC class II molecules.

The T cell induces IL-l on the macrophage, and thereaftera cavalcade of chemotactic factors promote granuloma genesis[2, 4]. IFN-'}' increases the expression of MHC class II molecules on macrophages, and activated macrophage receptorscarry a crystallizable fraction of IgG to potentiate their abilityto phagocytose [4]. The end result is the epithelioid granuloma,which progresses toward fibrosis under the impact of transforming growth factor and platelet-derived growth factor.

T cell subsets, macrophages, and other immune cells produceseveral cytokines, the amounts and types of which determinefurther subclassifications of granulomas. CD4+ T helper (Th)lymphocytes are necessary for the development of granulomasand are broadly classified into two functional types, Thl andTh2 [5]. Thl-type cells produce IL-2, IFN-'}', and TNF-,B uponstimulation with antigen and also participate in delayed-typehypersensitivity responses, while Th2-type cells make IL-4,IL-5, IL-6, IL-9, IL-I0, and IL-13, cytokines which are important for development of B cells and eosinophilia.

There appears to be a complex relationship between Thltype and Th2-type responses and infectious granulomas. Granulomas that synthesize predominantly Th2-type cytokines, suchas those that form in response to parasite ova, make only smallquantities of Thl-type cytokines chronically [6], whereas granulomas such as those of tuberculoid leprosy make large quantities of Thl-type cytokines and less of Th2-type lymphokines[7]. Granulomas of various infections may have different immunoregulatory mechanisms governing their formation andresolution [I, 2, 7].

Classification of Granulomatous Infections

Granulomatous disorders are frequently due to a wide varietyof infections. Over the past decade advances in molecular diag-

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Table 1. Classification of granulomatous infections and associatedpathogens.

Group 1 Granulomatous Infections (Causal AgentsWell Defined)

nostic techniques have allowed identification of organismsinvolved in granulomatous disorders that previously were ofunknown etiology. On the basis of currently available information, granulomas associated with infections can be categorizedin three groups, as listed in table 1.

Group 1 granulomatous disorders are caused by well-recognized agents. In group 2, the causal agents have been suspectedover the years but have only recently been recognized by newerdiagnostic molecular techniques such as PCR. These agents arenot readily isolated by conventional bacteriologic techniques.Group 3 comprises granulomatous disorders that may be associated with particular infectious agents but for which the causalorganisms have not yet been determined. Future advances indiagnostic techniques may provide strong evidence of thecausal agents, in which case the disorders may be reclassifiedas group 2; any such advancements will allow better management of these idiopathic granulomatoses.

Group number,type of causal agents

Group IWell recognized

Mycobacteria

Bacteria

SpirochetesFungiProtozoaNematodesTrematodes

Chlamydia species

Rickettsia

Viruses

Group 2Recently recognized

Bacterium

Actinomyces

Group 3Suspected but not established

Measles virusMycobacterium? (see table 4)

Viral

?

Granulomatous disorders

Tuberculosis, leprosy, Buruli ulcer,

swimming pool (fish tank)

granuloma

Brucellosis, melioidosis, actinomycosis,

nocardiosis, granuloma inguinale,listeriosis, tularemia

Syphilis, pinta, yawsMycoses (see table 3)Leishmaniasis, toxoplasmosisVisceral larva migrans (toxocariasis)Schistosomiasis, paragonimiasis,

fascioliasis, clonorchiasisLymphogranuloma venereum, trachoma

Q fever (Coxiella burnetii infection)

Infectious mononucleosis,cytomegalovirus infection, measles,

mumps

Cat-scratch disease (Bartonella

henselae infection)Whipple's disease (Tropheryma

whippelii infection)

Crohn's diseasePrimary biliary cirrhosis

SarcoidosisKikuchi's disease

Chronic granulomatous disease of

childhood

Langerhans granulomatosis

A wide range of infectious organisms cause the granulomatous diseases that fall into this category (table 1).These diseasesshare similar histologic features and have identifiable and distinct etiologic agents.

Mycobacterial Infections

Mycobacteria are a large group comprising Mycobacteriumtuberculosis, Mycobacterium leprae, Mycobacterium aviumcomplex, Mycobacterium ulcerans, Mycobacterium marinum.several opportunistic mycobacteria, and the mycobacteria fromwhich BCG vaccine is produced. The nontuberculous mycobacteria produce a wide clinical spectrum of granulomatousdisorders, summarized in table 2. Mycobacteria have been implicated in the etiopathogenesis of sarcoidosis and Crohn' sdisease, and this is discussed in the section on group 3 disorders.

Tuberculosis. The etiologic agent of tuberculosis, M. tuberculosis, is clinically one of the most common microbialcauses of granulomas [2, 8]. The M. tuberculosis granuloma,classically characterized by the presence of central caseousnecrosis, is referred to as a tubercle. Typically, there is a centralarea of amorphous caseating granular debris and loss of cellulardetail, and acid-fast bacilli are present. This area is encircledby epithelioid cells, lymphocytes, histiocytes, fibroblasts, andoccasionally Langhans' giant cells. These histologic featuresof the granuloma are sufficiently characteristic to allow reasonably accurate diagnosis of tuberculosis [8].

While caseating granulomas are the classic finding in casesof tuberculosis, they are not always present; noncaseating granulomas may occur. When acid-fast bacilli are not identifiablein the granuloma, culture of the biopsy specimen may yieldM. tuberculosis. PCR with use of primers specific for M. tuberculosis is currently under evaluation for the identification ofthis species [9] and may develop into an important tool fordistinguishing it from other (nontuberculous) mycobacteria.

Immunosuppression may alter the classic histopathologicfeatures of tuberculosis. A spectrum of histopathologic granulomatous tissue reactions are seen in patients infected with HIV[10]. Classic well-formed granulomas have been observed inindividuals with early HIV disease whose CD4 cell counts haveremained adequate. In patients with advanced HIV disease, thegranulomas are less well formed, are more necrotic, and maycontain abundant bacilli, which may be demonstrated by appropriate staining techniques as well as by culture of biopsy specimens [9-11].

Leprosy. Leprosy, or Hansen's disease, is a chronic granulomatous disorder caused by M. leprae, an obligate intracellularbacterium that predominantly affects the skin and nerves [12].First identified over a century ago by Armauer Hansen, thebacterium has defied all attempts to grow it in culture. Leprosy



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148 Zumla and James

Table 2. Granulomatous diseases caused by nontuberculous mycobacteria.

em 1996;23 (July)

Site

Lung

Lymph node

Bones and joints

Skin and soft tissue

Multisystem

involvement

Clinical manifestation

or type of disease

Bronchopulmonary disease

Lymphadenitis

Osteomyelitis

Abscesses

Buruli ulcer

Swimming pool granuloma

Tuberculoid leprosy

Lepromatous leprosy

Meningitis, diarrhea, pneumonia,

lymphadenitis

Mycobacterial species

M. avium complex, M. kansasii, M. xenopi,

M. simiae, M scrofulaceum, M. chelonae,

M. malmoense

M. avium-M. intracellulare, M kansasii,

M fortuitum, M. scrofulaceum,

M. chelonae

M. avium-M. intracellulare, M. kansasii,

M fortuitum, M scrofulaceum,

M. marinum

M chelonae, M. fortuitum

M. ulcerans

M. marinum

M. leprae

M. leprae

M. avium-M. intracellulare, M. kansasii,

M. chelonae

manifests as a spectrum of well-described clinical, pathological, and immunologic features [12].

At one end of the spectrum is lepromatous leprosy, the lowresistance form with multiple lesions, extensive skin and visceral involvement, and diffuse infiltration of lesions with numerous leprotic baccilli. At the other end of the spectrum,tuberculoid leprosy, the highly resistant form, is characterizedby one or few skin lesions and involvement of the nerve at thesite of the lesion. Skin biopsies of patients with tuberculoidleprosy reveal a few acid-fast and alcohol-fast leprotic bacilliand a well-defined giant cell granuloma. Actual granulomatousinvasion and destruction of dermal nerves, occasionally withcaseous necrosis, sometimes occurs.

Within the dermis of patients with lepromatous leprosy, increased levels of Th2-type cytokines (lL-4, IL-5, and IL-IO)and decreased levels of IL-2 and IFN-y have been noted. Incontrast, ThI-type cytokines predominate in tuberculoid lesions[7]. The diagnosis of leprosy is based on a combination ofclinical and histologic findings. The paucity of acid-fast bacilliin the lesions of tuberculoid leprosy may sometimes make itdifficult to distinguish them from other infectious and noninfectious granulomas, especially those of sarcoidosis and syphilis[12]. Pt.R for the detection of M leprae in tissue lesions isbeing developed, and this may in the future allow more accuratediagnosis in such circumstances [13].

Buruli ulcer. M. ulcerans is the cause of chronic, relativelypainless, cutaneous Buruli ulcers. The disease is most prevalentin Africa and Australia. The organism causes extensive undermined ulcers on the extensor surface of the extremities. Thecenters ofthe ulcers are necrotic, and the edges are undermined;the organisms are usually found at the periphery, where granulation tissue is most extensive [14]. While it is relatively easyto diagnose Buruli skin ulcers on the basis of clinical features

and histologic findings, microbiological identification of thecausal mycobacteria may sometimes be quite difficult, requiring long periods of culture.

Newer techniques such as gas-phase chromatography arebecoming useful for identification of the acid-fast bacilli inlow-count subcultures [15]. A report from West Africa [16] ofa case of disseminated M. ulcerans infection that followed asnakebite and caused multifocal osteomyelitis illustrates thedifficulties that are associated with identifying the organismby conventional microbiological techniques. Although repeatedcultures were negative, the organism was identified as M. ulcerans by peR amplification of the genes coding for 168 rRNAfrom biopsy specimens.

Swimming pool (fish tank) granuloma. M marinum causesswimming pool (fish tank) granuloma [17]. Although the primary skin infection may be inconspicuous, the draining lymphnodes are extensively involved and caseous. A similar microscopic picture, with conspicuous plasma cell infiltration, is associated with granulomas due to other opportunistic mycobacteria. Fish tank granulomas develop in persons with minorabrasions who dip their hands in tropical fish tanks. Usually asolitary granuloma, nodule, or pustule forms, which may ulcerate or suppurate; however, multiple lesions may extend alongthe line of lymphatic vessels (in sporotrichotic infections).

Biopsy specimens that are cultured on Lowenstein-Jensenmedium at room temperature yield pigmented mycobacterialcolonies in 2~4 weeks. The response to treatment is variableand not dramatic. Antituberculous drugs, cotrimoxazole, andhigh doses of minocycline have been advocated.

A wide range of mycobacterial diseases occur in humans(table 2), and in some cases the clinical features and results ofstaining for acid-fast bacilli in granulomas may not reveal theidentity of the causal organisms, which may also be difficult



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to isolate in culture. The development and application ofmolecular techniques such as PCR may in the future allow fairly

accurate diagnosis [18].

Bacterial Infections

Brucellosis. Brucellosis (Malta fever) is a multisystemgranulomatous disorder caused by small gram-negative, intracellular coccobacilli of the Brucella species [19]. The UnitedKingdom is one of 17 Brucella-free countries. It is more prevalent than its reported incidence in the United States, France,Spain, and Ireland [20]. Unpasteurized dairy products remaina common mode of transmission of this chronic granulomatousdisorder among travelers to areas of endemicity. Raw camel'sand goat's milk, raw sheep's and goat's liver, and reindeerbone marrow have all been associated with transmission.

Diagnosis, which may be difficult by means of culture, isusually based on clinical features and serological and histopathologic findings [20]. Delayed-type hypersensitivity is believedto be important in the formation of the tissue granuloma, whichlimits the spread of the organism [21]. Epithelioid-cell granulomas may be found in the reticuloendothelial system, lung, brain,bone, joints, and soft tissue and are difficult to distinguish from

granulomas due to other causes.Necrotizing hepatic granulomas due to brucellosis have been

described. In these cases, bacteriologic diagnosis may be difficult on the basis of blood or abscess pus cultures, althoughserology may be useful [22]. PCR tests for the identificationof Brucella species are being developed [23, 24], and thesemay be useful tools for the diagnosis of brucellar granulomas.

Melioidosis. Melioidosis is caused by a gram-negative bacillus, Burkholderia pseudomallei. Acute melioidosis presentswith protean clinical manifestations during which septicemia

may occur, causing septic shock and multiorgan involvement.This may progress to a chronic suppurative phase, in whichlung involvement, subcutaneous abscesses, lymphadenitis, andsuppurative parotitis are common [25, 26]. Granulomatous osteomyelitis has also been described [27].

The lesions vary histologically from acute, chronic inflammation with a focal granulomatous component to a predominantly granulomatous picture. Caseous or purulent central necrosis is surrounded by epithelioid and giant cells and byfibrosis in lungs, lymph nodes, and bone. The presence ofintracellular gram-negative bacteria within macrophages or giant cells and of similar extracellular bacilli may provide a clue

to the diagnosis [28].

More-specific enzyme immunoassays are being developedfor the detection of circulating antibody [29] or of B. pseudomallei antigen in the urine [30] of patients with melioidosis.PCR tests for the identification of B. pseudomallei have recently been described [31, 32], but these require field testingin areas of endemicity.

Treponemal Infections

The spirochetes include Treponema pallidum subspecies pal

lidum (the cause of syphilis), T. pallidum subspecies pertenue(responsible for yaws), and Treponema carateum (the causalagent of nonvenereal pinta in Latin America). All three arechronic granulomatous disorders that may cause diagnosticconfusion with other granulomatous disorders [33]. Mucocutaneous lesions of secondary syphilis show a spectrum ofhistopathologic changes, ranging from minimal infiltration togranulomatous infiltration throughout the dermis [34]; granulomatous infiltrates show endothelial proliferation, with mononuclear cell infiltration.

The syphilitic gumma may present as a microscopic togrossly visible lesion, with an enclosing wall of histiocytes,plasma cell infiltrate, and necrotic center cells with intact cellular outlines. peR to detect T. pallidum subspecies pallidumDNA in clinical specimens, including gumma biopsy material,appears to be highly sensitive and specific [35, 36].

Protozoan Infections

Leishmaniasis. Leishmaniasis refers to the spectrum ofclinical disease caused by the protozoan Leishmania [37]. Thisparasite is transmitted by phlebotomine sandflies, and the disease presents in three clinical forms: cutaneous, mucocutaneous, and visceral. Amastigotes of Leishmania species livewithin macrophages and are usually seen as single or focalcollections within macrophages in blood, aspirates of sternalmarrow, liver or spleen specimens, or special culture medium.

Leishmaniasis has been extensively studied as a model infection for analysis of cell-mediated immunity. Granulomatousresponses are a feature of cutaneous and mucocutaneous leishmaniasis. Cutaneous ulceration is characterized by a mononuclear cell infiltrate, with Th l-type responses emerging afterweeks of infection. Resolution of the infection depends on anincrease in the number of leishmania-specific CD4+ T cells ofthe Thl subset [38], following which a granulomatous responsewith epithelioid and giant cells emerges.

The nodular stage of post-kala-azar dermal leishmaniasis ischaracterized by massive granulomas consisting of lymphocytes, plasma cells, and histiocytes, with numerous amastigotesof the species Leishmania donovani [39]. A preponderance ofCD8+ T cells occurs in the lesion, in contrast to normal levelsin the peripheral blood. A recent report described the use ofPCR for identifying Leishmania aethiopica DNA in formalinfixed and paraffin-embedded tissue specimens [40]: seven ofthe 40 skin biopsy specimens in which parasites were not foundhistopathologically were subsequently found to contain parasite DNA.

Toxoplasmosis. Toxoplasmosis refers to the clinical disease caused by the protozoan Toxoplasma gondii. Neonatalinfection occurs during acute maternal infection in pregnancy.Acquired toxoplasmosis presents as a glandular, fever-like syn-



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150 Zumla and James

Table 3. Causes and immunopathologic features of granulomatous mycoses.

em 1996;23 (July)

Fungal species

Cryptococcus neoformans

Candida species

Sporothrix schenckii

Histoplasma capsulatum

varieties

Aspergillus speciesParacoccidioides brasiliensis

Coccidioides immitis

Blastomyces dermatitidis

Phialophora speciesPseudallescheria boydii,

Madurella species

Clinical condition

Cryptococcosis

Candidiasis

Sporotrichosis

Histoplasmosis

Aspergillosis

South American blastomycosis

Coccidioidomycosis

Blastomycosis

ChromoblastomycosisMycetoma

Immunopathologic feature(s)

Pneumonia, infarction, abscess,

meningitis, granuloma, fibrosisAbscess, necrosis, granulomas

(mucocutaneous or multisystem)

Granuloma (cutaneous, skeletal)Pneumonia, cavitation, granuloma

Necrotizing multisystem granulomasPneumonia, cavitation, granuloma

Pneumonia, cavitation, granuloma,

cutaneous plaques, nodulesGranuloma, microabscess, pneumonia,

chronic pulmonary or extrapulmonarydisease

Granuloma (cutaneous)Granuloma (skin and subcutaneous tissue)

drome that rapidly resolves. The T. gondii trophozoites encystand persist in nucleated cells as the cause of chronic, latentinfection that can reactivate with immunosuppression and produce multisystem disease.

The histopathologic changes characteristically occur with atriad of features [41]: reactive follicular hyperplasia, epithelioidhistiocytes, and monocytoid cells. Langhans' giant cells arenot typically seen. Accurate diagnosis may be difficult in manycases. PCR is increasingly being used to detect T. gondii DNAin various clinical samples [42], and its application to identification of T. gondii DNA in tissue biopsy specimens is underinvestigation.

Fungal Infections

Fungal infections are common causes of granulomas worldwide. Granulomatous fungal infections can present as localizedconditions or systemic illnesses (table 3). A wide range ofmedically important fungi can now be diagnosed by PCR [43].Histoplasmosis and coccidioidomycosis are caused by dimorphic fungi that produce clinical disease resembling tuberculosis[44-46].

Histoplasma infections in the lungs of immunocompetentpersons cause epithelioid-cell granulomas that undergo coagulative necrosis. Histologic differentiation from tuberculosis,sarcoidosis, and coccidioidomycosis may be possible by identification ofthin-walled yeast forms in Grocott-Gomori methenamine-silver nitrate stains. In immunodeficient persons, histoplasmosis may be fulminant and is not associated withformation of epithelioid-cell granulomas: focal collections ofmononuclear phagocytes containing yeasts are seen throughouttissues of the body, and the reticuloendothelial system becomesfull of macrophages containing yeasts [46, 47].

Coccidioides immitis spores inhaled by immunocompetentpersons induce a delayed-type hypersensitivity reaction. Mostprimary cases of coccidioidomycosis involving such patientsare asymptomatic. Lung lesions may develop in some patients,in association with fever, chest symptoms, and erythema nodosum. A minority of cases progress to disseminated infection.

The primary and secondary granulomatous lung lesions dueto C. immitis are similar to those due to Histoplasma species.C. immitis organisms are thick-walled, nonbudding spherulesoften filled with endopores. A neutrophil infiltrate may occuraround the area of granulomatous reaction when the spherulesrupture to release the endospores. In disseminated disease, theinflammatory response may be purely granulomatous, pyogenic, or mixed. Pyogenic lesions may dominate in immunosuppressed patients [46, 47].

Helminthic Infections

Several helminthic parasites can cause granulomas withpathological consequences. Ofparticular interest are schistosomiasis and visceral larva migrans.

Schistosomiasis. Schistosomiasis is caused by five mainhuman-blood flukes of the genus Schistosoma: S. haematobium,S. mansoni, S.japonicum, S. intercalatum, and S. mekongi.These currently infect more than 200 million people. Diagnosisis made by identification of the species-specific characteristicova in feces, urine, or tissues (usually rectal and liver biopsyspecimens). During the chronic phase of the infection, the mature worms produce large numbers of ova, which becometrapped in tissues and induce formation of granulomas [48],with chronic fibro-obstructive sequelae.

Granuloma formation is the result of a combination of factors: a foreign-body reaction to the deposited ova (which may



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or may not be identifiable within the granuloma) and a cellmediated delayed-type hypersensitivity reaction to antigenicdeterminants of the parasite [49]. The giant cells are of themultinucleate type, and the eosinophils are conspicuous. Theova contain living larvae that release a variety of toxic andantigenic substances through the egg wall, which itself is antigenic.

These substances induce and maintain the granulomatousresponse. The granulomatous response appears to be tightlyregulated and involves development of Th l-type and Th2-typecytokine responses [50, 51].

Toxocariasis (visceral larva migrans). Dog and cat larvalnematodes, especially Toxocara canis and Toxocara cati, mayinfect children and lead to the syndrome of visceral larva migrans [52]. Swallowed infected eggs emerge as larvae in theintestine, whence they pass to the liver, lungs, brain, and eye,producing eosinophilic granulomas in these different organs.This infection should be particularly suspected in children withhepatomegaly or miliary granulomas in liver biopsy specimens,miliary lung mottling, eosinophilia, and focal posterior uveitisor endophthalmitis. Liver granulomas may reveal part of thelarvae with surrounding eosinophilia.

Viral Infections

Measles. The paramyxovirus family is a group of RNAviruses that includes the measles virus, which has been implicated in the etiology of idiopathic granulomatous disorders,sarcoidosis, and Crohn's disease. The measles (rubeola) virusis spread by respiratory droplets and it multiplies within theupper respiratory tract epithelial cells, mononuclear cells, Band T lymphocytes, and macrophages. Viremia follows, whichdisseminates the virus throughout the body. T cell-mediatedimmunity that controls the infection develops and produces themeasles rash.

The blotchy rash of measles consists of dilated skin vessels,edema, and a nonspecific mononuclear perivascular infiltrate[47,53]. Lymphoid organs have marked follicular hyperplasia,large germinal centers, and randomly distributed multinucleategiant cells (Warthin-Finkeldey cells), which have eosinophilicnuclear and cytoplasmic inclusion bodies. In the lung, peribronchial and interstitial mononuclear infiltration occurs. The measles virus has been implicated in the etiopathogenesis of sarcoidosis and Crohn's disease and is discussed in the sectionon group 3 disorders.

Herpesviruses. The herpes group of viruses are doublestranded DNA viruses that cause a spectrum of human disease.The Epstein-Barr virus is the etiologic agent of the clinicalcondition infectious mononucleosis and is transmitted by closecontact, especially via saliva during kissing. It has also beenimplicated in the pathogenesis of several disorders such asBurkitt's lymphoma, nasopharyngeal carcinoma, B celllymphomas, and sarcoidosis.

Infectious mononucleosis in its benign form is characterizedby fever, sore throat, lymphadenopathy, splenomegaly, and theappearance of atypical activated T cells in the peripheral blood.Further multisystem involvement may occur, leading to hepatitis, meningoencephalitis, and pneumonitis. Atypical lymphocytes are seen in the portal areas and sinusoids of liver biopsyspecimens, and scattered isolated areas of focal parenchymalnecrosis filled with lymphocytes may occur [47].

This histologic picture is difficult to distinguish from that ofother types of viral hepatitis, such as hepatitis B, and from thatof cytomegalovirus [54] infections. Specific diagnosis can bemade by culture of the virus or identification of virus-specificDNA by PCR [55].

Group 2 Granulomatous Disorders (Recently RecognizedCausal Agents)

Cat-Scratch Disease

Cat-scratch disease or fever is also known as benign lymphoreticulosis or regional granulomatous lymphadenitis [56]. Itonly occurs in humans, especially those who are scratchedor bitten by kittens and then develop regional lymphadenitisproximal to the site of injury. Primary involvement is that ofthe lymph nodes, which first show lymphoid hyperplasia. Later,scattered granulomas with central areas of necrosis coalesce toform abscesses.

The histopathologic features of cat-scratch disease are notdiagnostic and may be mistaken for tularemia, lymphogranuloma venereum, syphilis, brucellosis, atypical mycobacterialinfections, fungal infections, and toxoplasmosis [47]. WarthinStarry silver staining is used to detect Bartonella henselae,which may be present in the early phase of the disease. A skintest antigen has been made from lymph node pus. It is inoculated intradermally, and the degree of induration and erythemais measured at 48 hours.

The cat-scratch antigen skin test is positive for about 90%of patients who are clinically suspected of having the disease.This test will become redundant when techniques for amplifying specific nucleotide sequences with peR come into general use. There is no well-recognized response to antibiotics,and recovery usually occurs without treatment.

Until recently, the causal agent remained controversial. Twoorganisms, Afipia felis and B. henselae, have been identifiedas potential candidates [57]. Substantial evidence is now accumulating that B. henselae, a small pleomorphic gram-negativebacillus, is the etiologic agent [58-60].

A PCR hybridization assay designed to amplify DNA fromB. henselae and A. felis was used on pus aspirates from 89skin test-positive patients with cat-scratch disease. B. henselaeDNA was found in 96% of samples, while no samples containedA.felis DNA [58]. An indirect fluorescent antibody assayfor B. henselae-specific antibody has been described thatappears to be sensitive for the diagnosis of cat-scratch disease [61].



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152 Zumla and James em 1996;23 (July)

Whipple's Disease

George Hoyt Whipple of Johns Hopkins Hospital initiallydescribed a 37-year-old missionary who presented with fever,polyarthritis, and steatorrhea. His report was entitled "A Hith

erto Undescribed Disease Characterized Anatomically by Deposits of Fat and Fatty Acids in the Intestinal and Mesenteric

Lymphatic Tissues" [62]. Whipple's disease is a chronicmultisystem granulomatous disorder that affects middle-agedwhite males. It presents, as did Whipple's patient, with fever,polyarthritis, weight loss, and diarrhea progressing to malabsorption.

Hepatosplenomegaly and generalized lymphadenopathy maybe present, and biopsy of the lymph node, liver, or small intestine will reveal sarcoid granulomas, of which PAS stains will

reveal foamy macrophages. The PAS-positive material corre

sponds with lysosomes containing bacilliform bodies. Electron

microscopy reveals rod-shaped bacilli, termed Whipple bacilli[63], Whipple's associated bacterial organisms, or (more correctly) Tropheryma whippelii [64-66].

The nucleic acids extracted from an endoscopic biopsy specimen of the proximal small bowel of a patient with Whipple'sdisease were subjected to amplification and nucleotide sequencing by PCR. The resulting PCR product from the bacterial 16SrRNA was then the subject of a computer database search forthe rRNA sequences most similar to it.

T. whippelii is an actinomycete that behaves like an intracel

lular pathogen and responds particularly well to antibiotics thatenter cells easily. It is an erythrocyte-associated organism (likeBartonella bacilliformis, Babesia species, and Plasmodiumspecies, which are also hemotropic). T. whippelii DNA has

been detected by PCR examination of peripheral blood, pleuraleffusion cells [66, 67], biopsy tissue from the intestine [68],heart [69], or vitrectomy specimen of the eye [70].

The PCR primers that have been developed are specific forT. whippelii, and this technique is now one of the standardmethods for establishing the diagnosis of Whipple's disease[66, 67, 70]. The technique ensures a more specific diagnosissince the organism cannot be cultivated. Other data show thatthere may be a closely related second causative agent of Whip

ple's disease [71, 72].

Group 3 Granulomatous Disorders (Infective AgentStrongly Suspected)

Primary Biliary Cirrhosis

This condition of progressive, nonsuppurative, granulomatous destruction of intrahepatic bile ducts has also been termedxanthomatous biliary cirrhosis, but its most accurate descriptionis chronic nonsuppurative destructive cholangitis [73]. Livercell necrosis may become superadded, later progressing to cirrhosis. Epithelioid granulomas associated with the bile ductsare found in about one-third of the cases of primary biliarycirrhosis (PBC). The condition has been likened to chronic

graft-versus-host rejection, with similar structural changes in

the bile, lacrimal, and pancreatic ducts, which have a highconcentration of human leukocyte class II antigens on the epithelial surface.

PBC is distinguished by the fact that it predominates in

women in the reproductive years of age and by the presenceof serum mitochondrial antibodies. It is classified as an autoim

mune disorder and is associated with other autoimmune conditions, including rheumatoid arthritis, systemic lupus erythematosus, scleroderma, Sjogren's syndrome, and the CRESTsyndrome.

The etiology of PBC remains unknown. Two infectiousagents have been postulated as possible trigger cofactors [74,75]. The antigen specific for PBC serum is M2, a component

of the pyruvate dehydrogenase complex of mitochondrial en

zymes. There are similarities between mitochondrial components and Escherichia coli, and cross-reactivity between bile

duct mitochondria and bacteria may be conceivable [73]. Mitochondrial antibodies cross-react with subcellular constituentsor gram-negative intestinal or urinary tract organisms.

An increased incidence of gram-negative urinary tract infections in patients with PBC has been reported. Another infectiveassociation was suggested in a report from Barcelona that incriminated Mycobacterium gordonae. This ubiquitous organism causes granuloma formation, and serum antibodies are evident in PBC serum. Furthermore, there is cross-reactivity

between this organism and the important PBC-defining M2

antigen. PCR has disclosed the presence of this mycobacteriumin the tissues and bile of patients with PBC [75].

Sarcoidosis

Sarcoidosis is a multisystem disorder of unknown etiology[76]. An antigen (chemical or infectious organism)-driven,cell-mediated immune response leads to a cytokine cascade, tosarcoid granuloma formation, and eventually to sarcoid fibrosis.An intense search for an infectious cause has yielded manytheories, yet none is conclusive (table 4).

In 1961, Edith Mankiewicz [77], of Montreal, reported thatbacteriophages, lytic for mycobacteria, can be isolated withhigher frequency from stool and resection specimens from pa

tients with tuberculosis and sarcoidosis. Raised titers ofphageneutralizing antibodies were found in tuberculous patients infected with mycobacteria harboring mycobacteriophages butnot in cases of sarcoidosis.

While mycobacteriophages are common to both diseases

the persistence ofphages without antibody was the explanationgiven for the absence of M. tuberculosis in sarcoid tissue [78].This observation led to further experiments in which guineapigs were infected with tubercle bacilli alone or together withmycobacteriophages, and the histological responses (analyzedblindly) were of two dissimilar types. The first was caseousnecrosis with the presence of acid-fast bacilli, and the secondwas a different response modified by the mycobacteriophages:



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cm 1996; 23 (July) Granulomatous Infections 153

Table 4. Causal agents implicated in cases of sarcoidosis.

the phage-infected animals showed a granulomatous sarcoidlike picture with very few atypical lysogenic mycobacteria.

The conclusion reached was that phages of different antigenic composition determined a shift in the antigen-antibodyresponse toward either caseation or noncaseating granulomaformation. It seemed at that time that sarcoidosis resulted fromunrestrained lytic phage activity against mycobacteria [77, 78].

At about the same time, Chapman and Speight [79] reporteda high incidence of serum antibodies to mycobacteria in patients with sarcoidosis. Serum antibody levels were particularlyhigh against photochromogen and scotochromogen antigens.The patients' antibody status changed from negative to positiveas sarcoidosis manifested itself, and in many cases antibodiesappeared 2 months following the onset of erythema nodosum.

Ghosts of mycobacteria have been reported to be detectedby fluorescent microscopy of scalene lymph node biopsy specimens from patients with sarcoidosis and from controls. Auramine-rhodamine stains disclosed shining golden rods resembling mycobacteria in specimens from patients with sarcoidosisbut not in those from controls [80]. Recent advances in molecular biology have resurrected these observations; PCR has detected mycobacterial DNA in clinical samples from patientswith sarcoidosis [81, 82].

Bronchoalveolar lavage samples, bronchial washings, andtissue specimens have been assayed by PCR for mycobacterialDNA. M. tuberculosis DNA has been found in 50% of patientswith sarcoidosis, and nontuberculous mycobacterial DNA hasbeen found in 70% of them. Another study extracted RNAfrom five sarcoid and five normal spleens [81]. Extracts wereassayed by liquid-phase DNA/RNA hybridization with a DNAprobe specific for the rRNA of the M. tuberculosis complex.

Causal agents implicated

Bacteria

Mycobacteria

Streptococci

Propionibacterium acnes

Borrelia burgdorferi

Mycoplasma species

Nocardia species

Viruses

Epstein-Barr

Herpesvirus

Rubella

Measles

Cytomegalovirus

Coxsackievirus B

Retrovirus

Others

Beryllium

Zirconium

Pine pollen

Peanut dust

Clay (ingestion)

Reference(s)

[76-92]

[94]

[88,93]

[95,96]

[97]

[98]

[99]

[100, 101]

[102]

[102]

[102]

[102,103]

[104]

[lOS]

[105][106,107]

[107]

[108]

The number of hybrids obtained with the sarcoid spleens (fromwhich mycobacteria were neither seen on microscopy nor cultured) was 4.8 times higher than that obtained with normalspleens.

However, the results obtained by PCR are being fiercelycontested by German [83], Scottish [84], and French [85] investigators. The findings of the German group contradict those ofSaboor et a1. [81], and the group concludes that mycobacterialDNA cannot be detected in tissues or bronchoalveolar lavagecells in cases of sarcoidosis. They use the technique to distinguish tuberculosis (which produces a positive result) from sarcoidosis. The Glasgow investigators [84] also failed to detectmycobacterial DNA in sarcoid lymph nodes. The French investigators [85], using PCR, rarely found DNA from M. tuberculo

sis in cases of sarcoidosis.The literature for and against a mycobacterial cause of sar

coidosis is summarized in table 5. The existence ofmycobacterial sarcoidosis would validate several claims of infective orchemical causes of sarcoidosis: Scadding's long-held views ontuberculous causation [86], a Swedish suggestion [87] of aninteraction between mycobacteria and a virus, a Japanese theory [88] regarding Propionibacterium as the causal agent, andthat of investigators in Houston [89] who isolated cell walldefective spheroplasts of acid-fast mycobacteria from sarcoidskin biopsy specimens.

The controversy continues [90-93] and many different antigens have come under suspicion, including other bacteria [9497], fungi [98], viruses [99-104], and chemicals [105-- 108].Many attempts have been made but tissue culture has failed touncover a virus. Elevated antibody responses to several viruseshave been reported, but such responses may only reflect B-cellhyperreactivity rather than a causal relationship.

Crohn's Disease

Crohn's disease is a chronic granulomatous disorder affecting predominantly the gut, but it has multisystem involvementand variable clinical manifestations. The pathological changesmay involve any parts and any layer of the alimentary tract.Transmural inflammation consists of chronic inflammatorycells with lymphoid aggregates scattered throughout. Noncaseating sarcoid-like granulomas may be present in all layers ofdiseased and unaffected tissue throughout the alimentarytract [109].

As with sarcoidosis, controversy prevails concerning a causalagent. Clinicopathologic studies at the Royal Free Hospital(London) demonstrated the presence of granulomatous vasculitis and vessel thrombosis, giving rise to focal microulcers. Theinvestigations have shown early superficial mucosal changesand disruption of the capillary basement membrane precedingthe formation of the microulcer [I 10]. These inflammatorychanges may extend beyond the alimentary tract to the lung,causing pulmonary vasculitis, granulomatous interstitial lymphocyte infiltration, and interstitial fibrosis [II I, 112].



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154

Table 5.

Reference( s)

[77,78]

[79]

[80]

[87]

[93]

[89]

[81]

[82]

[83]

[84]

[85]

[90]

[91]

[92]

Zumla and James

Summary of the literature for and against a mycobacterial cause of sarcoidosis.

Laboratory method used or featureYear(s) of study Location of study analyzed (finding)

1961-1964 Montreal Reaction to mycobacteriophage infection1964 Dallas Presence of mycobacterial antibodies in

serum (positive)

1971 Prague Auramine-rhodamine staining,

fluorescent microscopy (positive)

1972 Stockholm Mycobacteria-virus interaction

1984 Tokyo Culture of lymph nodes (positive for

Propionibacterium acnes)1988 Houston Skin biopsy (cell-wall-defective, acid-

fast bacilli noted)1992 London PCR (positive for mycobacterial DNA)

1992 London Liquid-phase hybridization (positive for

mycobacterial rRNA)1992 Borstel, Germany PCR (negative for mycobacterial DNA)

1992 Glasgow PCR (negative for mycobacterial DNA)

1992 Paris PCR (negative for mycobacterial DNA)

1993 London PCR (positive for mycobacterial DNA)

1993 Ohio ELISA (positive for antibodies to

Mycobacterium paratuberculosis)1993 Copenhagen Western blotting (positive for antibodies

to Mycobacterium tuberculosis)

ern 1996;23 (July)

Against this background is the conflict about whether thecausal agent is Mycobacterium paratuberculosis [113], othermycobacteria [114], or the measles virus [115, 116]. Measlesvirus nucleocapsids have been detected in foci of granulomatous inflammation of the intestine in cases of Crohn's disease[117]. It has been postulated that Crohn' s disease may be aresult of chronic granulomatous vasculitis that develops in reaction to a persistent infection with measles virus within thevascular endothelium [118].

Langerhans' Cell Granulomatosis

The term Langerhans' cell granulomatosis refers to proliferative disorders of histiocytes, previously referred to as histiocytosis X [119]. It encompasses a group of disorders of unknown etiology characterized by granulomatous infiltration ofthe lungs, bone, skin, lymph nodes, and brain. The clinicalconditions have been known by several names, based on thetype of presentation, sites of involvement, rate of progression,and degree of associated immune dysfunction. They includeeosinophilic granuloma, Letterer-Siwe disease, and HandSchuller-Christian disease. These are now considered to bedifferent expressions for the same basic disorder, in which theproliferation of Langerhans' cells results from disturbances inimmunoregu1ation.

Langerhans' histiocytes are bone marrow-derived monocyte-macrophage cells; they include Langerhans' epidermalcells, Kupffer's cells in the liver, osteoclasts, and alveolar macrophages. They are human leukocyte antigen-DR-positive

functioning macrophages that present antigen to T cells andplay a role in cell-mediated immunity. Unlike histiocytes,Langerhans' cells can be stained immunohistochemically forS-100 protein and OKT-6.

Lung biopsy reveals a mixed celullar exudate, foam cells,eosinophils, and characteristic X bodies (Birbeck granules) inmacrophages. Langerhans' or X bodies are an ultrastructuralfeature in 90% of patients [120]. They are identical to thegranules in Langerhans' epidermal cells and consist of intracytoplasmic rod-, plate-, or cup-like pentalaminar structures.

The presence of these tennis racket-shaped ultrastructuralBirbeck granules is diagnostic of the disorder. They have surface adenosine triphosphate activity identifiable by gold fluorescence. These diagnostic cells are readily found by bronchoalveolar lavage, and this technique may make lung biopsyunnecessary. It may also be a likely means of detecting apossible causal agent.

Chronic Granulomatous Disease of Childhood (CGD)

CGD comprises a genetically heterogeneous group of diseases that have in common the functional disorder of a multicomponent enzyme system, NADPH oxidase, found in phagocytic cells [121-124]. This defect leads to severe infections,particularly with Staphyloccocus aureus, Serratia marcescens,Burkholderia cepacia, and Aspergillus species. Organisms thatlack catalase supply the neutrophil with the hydrogen peroxidefor their own destruction. Thus, catalase-negative organismssuch as pneumococci or streptococci present no problem because they are normally destroyed.



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There are three autosomal recessive types, corresponding tomutations in p22p h0X, p47ph0X, and p67phox

. The classic X-linkeddisorder (found in ,....,60% of cases), which affects the geneencoding gp91phox, occurs in boys and is diagnosed in infancy,usually on the basis of an infection with a catalase-producingbacterium or fungus. The diagnosis is confirmed on the basisof a nitroblue tetrazolium test or other test showing defectivesuperoxide production.

Hepatosplenomegaly is common and does not reflect activeinfection per se. Lymphadenopathy is often undetected, butwhen it is significant it usually reflects an active infection.Severe granulomatous problems can occur in association withCGD, such as obstruction of the gut and the genitourinarysystem. Weeping granulomatous skin lesions occur in the setting of an active wound, typically post-surgically. The histological appearance is marked by lipid-laden macrophages. Whilethe etiology of CGD is genetic, there may be undetected pathogens responsible for some of the granulomatous complicationsof the disease.

Orofacial Granulomatosis (Melkersson-Rosenthal Syndrome)

This is a rare granulomatous disorder of the mouth andadjacent tissues, involving the oral mucosa, gum, lips, tongue,pharynx, eyelids, and skin of the face. Melkersson [125] described an association between facial edema and facial paralysis. Rosenthal [126] added the features of lingua plicata orscrotal tongue. Other clinical features include granulomatouscheilitis, edema of the gums and scalp, salivary gland dysfunction, granulomatous blepharitis, trigeminal neuralgia, Raynaud's phenomenon, and chronic hypertrophic granulomatousvulvitis [127-129].

Biopsy of an involved area reveals a noncaseating granuloma. Unlike the findings in cases of sarcoidosis, there are nochest radiographic changes or uveitis, and the Kveim-Siltzbachskin test is negative. The etiology remains unknown.

Kikuchi's Disease

This disorder was described in 1972 by a Japanese pathologist and is characterized by lymphadenitis with focal reticulumcell hyperplasia, nuclear debris, and phagocytosis [130]. Thelymphadenitis may be proliferative, necrotizing, or xanthomatous. Immunohistologic analysis shows that the predominantcells involved in the lymphadenitis are various types of histiocytes, plasmacytoid monocytes, and T cells; B cells are absent[131]. Clinically there is localized, tender, cervicallymphadenopathy with an upper respiratory tract prodrome.

Kikuchi's disease was initially thought to occur mostly inwomen under the age of 30 years, although a recent study of79 cases showed that the preponderance of cases involvingfemales is not striking [131]. The disease runs a self-limitingcourse and is associated with a recurrence rate of3%. Kikuchi'sdisease has been recognized as a presenting feature of mixed

connective-tissue disease and has been noted in associationwith adult Still's disease and systemic lupus erythematosus, afinding leading to the hypothesis that Kikuchi's disease andautoimmune rheumatic disorders may share a common etiology [132].

The disease occurs worldwide and has often been confusedwith toxoplasmosis, cat-scratch disease, tuberculosis, infectiousmononucleosis, and non-Hodgkin's lymphoma. The cause ofthe disease is not known. A viral etiology is strongly suspectedon the basis of clinical features, although serological andultrastructural studies have not yet identified an infectiousagent [133].

Conclusions

Classification schemes are most useful when they provideinsight into the purpose of events and the etiologic mechanismsthat govern them. Granulomas are remarkably complex inflammatory foci that sequester organisms or other substancesresistant to degradation. While a large number of granulomatous disorders are recognized, infections are clearly the mostcommon underlying causes of granulomas in general.

It is apparent that granulomatous conditions ofdiverse etiologies share common histologic features, although the etiologicagent is not always identifiable. Although the histopathologicpatterns in various infectious granulomas may be sufficientlydifferent to prevent an accurate diagnosis, atypical presentations may necessitate identification of the specific etiologicagent by direct microscopic examination, culture, serology, ormolecular detection.

In several granulomatous diseases the etiologic agent is difficult to identify by microscopic examination, culture, or serological means. The availability of PCR for the diagnosis ofseveral infectious conditions has been of major importance indetecting the etiologic agents of granulomatous disease conditions that previously were considered to be of unknown etiology. In particular, PCR has been used successfully for detectingthe bacteriologic causes of Whipple's disease and cat-scratchdisease.

Further applications of molecular techniques may pin downthe microbiological etiology (if any) of several granulomatousdisorders of group 3 (table I), of which the etiology remainselusive.

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