Pathology of human immunodeficiency virus infection: Infectious conditions

REVIEW ARTICLE

Pathology of Human Immunodeficiency Virus Infection: Infectious Conditions Michael R. Lewin-Smith, MB, BS, Mary K. Klassen, MD, Sarah S. Frankel, MD, and Ann Marie Nelson, MD Infection with the human i m m u n o d e f i c i e n c y virus (HIV) and the subsequent derangement of host immunity place affected patients at risk for secondary infections. Some of the secondary pathogens occur with such frequency or are so rare in the n o n - i m m u n o s u p p r e s s e d population that they have b e c o m e part of the Centers for Disease Control and Prevention (CDC) classification for HIV/ acquired immune deficiency syndrome (AIDS). Other infectious agents not yet included in the CDC definition are being reported in the HIV-infected population with increased frequency. General observations of the degree of immunosuppression associated with specific secondary infections have been useful in developing classification systems for HIV disease such as that of the CDC. However, the specific alterations in host immunity that promote infection with specific secondary pathogens are generally unknown. Geographic differences in the types and frequency of secondary infections also have been reported. Variation in strains of HIV, effect of malnutrition, lack of appropriate medical treatment, prevalence of virulent infectious diseases, and epidemiologic differences are possible contributing factors. Some infections that s e e m e d likely to be closely associated with HIV infection have not occurred more frequently in HIV-infected patients. This review summarizes the histopathology of infectious conditions in the current CDC classification and highlights s o m e conditions seen in HIV-infected individuals that are not currently HIV/AIDS-defining infections, yet may be seen by practicing pathologists. A n n D i a g n P a t h o l 2: 181-194, 1998. T h i s is a U S g o v e r n m e n t w o r k . T h e r e a r e no r e s t r i c t i o n s on its use.

Index Words: HIV, AIDS, infectious complications, pathology

T ~ P r E CENTERS FOR DISEASE CONTROL and evention (CDC) Classification System for HIV Infection I lists conditions used for surveillance. The majority of these conditions are infections. Human immunodeficiency virus (H1V)-infected patients present with uncommon infectious and common lesions in atypical sites or with atypical host reactions. An etiologic agent is often difficult to identify because of extensive antimicrobial therapy. Histologic evidence of multiple

From the Division of AIDS Pathology and Emerging I@ctious Diseases, Department of Infections and Parasitic DiseasePathology,Armed ForcesInstitute ofPathoIogy, Washington, DC. The opinions or assertionscontainedherein are the private views of the authors and are not to be construedas o~cia[ or as ~evting the views of the United States Department ofArmy or the Department of Defense or the institutions with which the authors are affdiated. Address correspondenceto Michael R. Lewin-Smith, MB, BS, 14th St and Alaska Ave, NW, Washington, DC 20306-6000. This is a US government work. There are no restrictionson its use. 1092-9134/98/0203-0006500.00/0

simultaneous co-infections may be present even in small biopsy specimens from a single lesion. Identification of different pathogens may require a panel of histochemical and immunohistochemical stains and other studies. The surgical pathologist continues to provide important information on the changing spectrum of complications of H1V infection and in the timely diagnosis of treatable conditions. Absolute CD4+ lymphocyte counts of less than 200 cells//xL are now considered to be diagnostic of the acquired immunodeficiency syndrome (AIDS)5 In addition to quantitative defects, CD4+ lymphocyte helper/ inducer functions are also abnormal with loss of the ability to respond to soluble antigens. A switch from TH1- to TH2-type cell-mediated immunity correlates with the progression to AIDS. TH1 CD4+ lymphocytes activate macrophages and promote the proliferation of other T lymphocytes. TH2 CD4+ lymphocytes promote proliferation of B cells and induce immunoglobulin isotype switching2

Annals of Diagnostic Pathology, Vol 2, No 3 (June), 1998: pp 181-194

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The monocyte/macrophage system functions of chemotaxis, phagocytosis, intracellular killing, and antigen presentation are variably reported as normal or defective in HIV-infected persons. Intrinsic function appears to be intact, but lymphokine-dependent function is abnormal. Because intact cell-mediated immunity is essential for granuloma formation, this response is affected by loss of TH1 function. Granulocytopenia increases both the risk and severity of bacterial and fungal infections. Hyperstimulated B cells are unable to respond to new antigens or to mount adequate immune responses, and susceptibility to infections by encapsulated organisms such as Streptococcuspneumoniae,Haemophilus influenzae, and Klebsiella sp is increased. The use of chemoprophylaxis has changed the incidence and spectrum of opportunistic infections. Multidrug antiviral therapy can result in increased CD4+ counts and restoration of the immune response.

vascular tumor. Small capillaries often ring ectatic vessels (Fig 1A). There is a collagen network with necrobiosis and overlying granular masses. The superficial portions often have stromal edema; deeper regions are cellular. In papular or nodular lesions, the overlying epidermis is thin, often with a collarette; ulceration with acute inflammation and bleeding may occur at this stage. Resolving lesions (rarely biopsied) are hyalinized and have fibroconnective proliferation rather than vascular proliferation.5 Parenchymal lesions of liver, spleen, and lung present as abortive granulomas, fibrosis (similar to that in skin), and bacillary peliosis. Histologically, peliotic lesions are large blood-filled spaces surrounded by a myxoid network of collagen with mixed inflammatory cells and karyorrhectic debris in the interstitium. Macrophages may be large with foamy cytoplasm and phagocytized cellular debris. Neutrophils are less common in peliosis than in other lesions.

Indicator Conditions

Bacteria Bartonellosis (bacillary angiomatosis/cat-scratch disease). Bacillary angiomatosis is an infection caused by the pleomorphic bacilliform bacterium, Bartonella henselae (or occasionally by Bartonella quintana or Afipia fells). Trauma to epidermal or mucosal surfaces is the usual route of transmission. Although not an AIDS indicator, most cases of bacillary angiomatosis occur in persons with a CD4+ T-cell count of less than 200 cells/b~L) After multiplication at the portal of entry, the organisms go to draining regional lymph nodes and viscera where they grow before sending multiple satellite colonies to random target areas of skin or mucosa. Cutaneous involvement is the most common presentation of bacillary angiomatosis, but it should be assumed that all infections are systemic. Visceral involvement is common; lesions have been reported in cardiac, respiratory, gastrointestinal, musculoskeletal, reticuloendothelial, soft tissue, and central nervous systems. Bone lesions are found in 35% of cases, often underlying an adjacent skin lesion. 4,5 The three main histologic stages are early granulomatoid, intermediate angiomatoid, and late hyalinized. Early granulomatoid (rarely biopsied) lesions show epithelioid and foamy macrophages, karyorrhexis, and necrobiosis of collagen. Intermediate angiomatoid lesions are the most frequently biopsied. Diagnostic features include a lobular vascular proliferation, with ectasia and protuberant endothelial cells mimicking a benign

Figure 1. (A) Bacillary angiomatosis. Note the proliferation of small vessels with prominent endothelial cells and blue-gray "clouds" around the vessels (Hematoxylin-eosin stain; original magnification x250). (B) Bacillary angiomatosis; clumps of bacilli appearing black with silver impregnation. (Warthin-Starry stain; original magnification x250.)

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Bacteria are abundant in early and intermediate stages in the necrobiotic areas, between collagen fibers, in the walls of vessels, or surrounding the peliotic spaces. The bacteria appear as granular "clouds" on most stains and as tangled masses with silver impregnation. When stained, the bacteria appear blue-graywith hematoxylineosin, red with Brown-Hopps gram stain, blue with Giemsa, and black with Warthin-Starry or WengerAngritt (Fig 1B). They are not seen or only rarely seen with the Grocott methenamine silver (GMS) and Steiner stains. Listeriosis. Listeria mono®togenes is a gram-positive, non-spore-forming, nonencapsulated, facultatively anaerobic, nonbranching bacillus. It has a worldwide distribution in humans, animals, soil, and contaminated foods. 6 Although uncommon in HIV-infected patients, the incidence of listeriosis is 65 to 145 times greater than in the general population. 7 The disease has similar manifestations in patients with and without HIV infection. In most cases, the source of infection, mode of transmission, and portal of entry are unknown. Bacteremia occurs in the majority of cases, often with concomitant meningitis. Lung, brain (meningitis or abscesses), heart (pericarditis or epicarditis), liver, and skin lesions also have been reported. The lesions show necrosis, degenerating neutrophils, and small, gram-positive, nonspore-forming bacteria. Cultures, especially blood or cerebral spinal fluid, are used more frequently to diagnose this infection than biopsy]

Pelvic inflammatory disease complicated by tuboovarian abscess. Pelvic inflammatory disease, including endometritis, salpingitis, pelvic peritonitis, and tuboovarian abscess, tends to be more frequent and more severe in HIV-seropositive women. Immunosuppressed women with pelvic inflammatory disease often require hospitalization and surgical intervention. Neisseria gonorrhoeae, Chlamydia trachomatis, aerobic and anaerobic streptococci, and other bacteria are the most common causes. No specific microbiologic or histopathologic differences have been reported in HIV-infected women, a Recurrent pneumonia. Recurrent pneumonia may be due to a single infection or bouts of different infections. Frequent antibiotic therapy and long-term prophylaxis have led to an increase in drug-resistant strains of bacteria. Cases of recurrent pneumonia in HIV-infected patients for whom culture data are available include the encapsulated bacteria (S. pneumoniae, H. inJluenzae, and Klebsiella sp), as well as other usual and unusual pathogens (eg, Chlamydia sp, Mycoplasma sp, Legionella sp, Moraxella catarrhalis,Rhodococcusequi, group B streptococcus, Staphylococcus aureus, Bordetella bronchisep-

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tica, BranhameUa catarrhalis, Pseudomonas aeruginosa, Enterobacter cloacae, Salmonella enteritidis, and mycobacterial, fungal, protozoal, and viral infections).6.9 Bacterial pneumonias represent an important cause of early mortality, are often multilobar, and are associated with significant bacteremia. Bacteria may enter the respiratory tract by aspiration or hematogenously. In most cases, histology shows a suppurative reaction, and organisms can be seen on tissue gram stain (BrownHopps), often within neutrophils. Mycoplasmafermentans may cause mild focal changes, including patchy necrosis with little inflammation, or rarely, fulminant infection with extensive infarction-like necrosis. 6 Mycoplasmal and chlamydial infections often require immunodiagnosis or electron microscopy for confirmation. Cases of pneumonia should be evaluated for multiple infections using a battery of special stains (ie, Brown-Hopps, GMS, acid fast, and, if needed, Warthin-Starry or other silver impregnation procedures).

Mycobacteria Mycobacteriosis (Mycobacterium avium complex disease, Mycobacterium kansasii), disseminated or extrapulmonary. Disseminated M avium complex (MAC) has a prevalence of approximately 22% in AIDS patients. More than 80% of cases occur after another AIDS-indicating condition, and the median CD4+ cell count among persons with MAC is 10 cells//zL. ~°,11The percentage of AIDS patients with disseminated MAC has increased. Infection often begins in the intestinal tract with proliferation of mycobacteria in macrophages of the lamina propria. As infection progresses, sheets of foamy macrophages fill and expand the villi, sometimes evolving into a Whipple-like process. 12 Mesenteric adenopathy usually occurs before dissemination of the infection. Lymph node involvement varies from tiny loci of infection to marked lymphadenopathy, with replacement of the normal architecture by sheets of foamy macrophages containing masses of mycobacteria. Other organs show clusters of foamy macrophages or poorly formed granulomas. These granulomas contain admixtures of foamy macrophages, lymphocytes, and occasionally a few epithelioid macrophages or polymorphonuclear leukocytes. Langhans giant cells and central caseous necrosis are extremely uncommon. Mycobacterial spindle-cell lesions similar to histoid leprosy have been reported with MAC infection; the spindled macrophages contain large numbers of bacilli. 13 In hematoxylin-eosin-stained tissue or in Giemsastained imprints or smears, the mycobacteria are nega-

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tively stained within the cytoplasm, giving the macrophages a striated appearance. Acid-fast, periodic acidSchiff (PAS), and silver impregnation techniques reveal masses ofintracytoplasmic bacilli.

Myeobacteriosis ( M y c o b a c t e r i u m t u b e r c u l o s i s ) , pulmonary or extrapulmonary. Patients with HIV infection are uniquely susceptible to both primary and reactivation disease caused b y M tuberculosis. Because M tuberculosis is a virulent pathogen, it is often the earliest infection in the course of declining immunocompetence. Tuberculosis itself is immunosuppressive and causes a depression in blood CD4+ lymphocyte counts in both HIV-infected and non-infected persons.14 The histologic patterns of tuberculosis reflect the integrity of the patient's cellular immune response. Patients with relatively intact cellular immunity have a typical granulomatous response with few organisms. As the CD4+ lymphocyte count decreases, cellular immunity decreases, and there is a loss of Langhans giant cells and a decrease in epithelioid rnacrophages. Necrosis is both suppurative and caseous. In this hyporeactive stage, there is poor intracellular killing of mycobacteria. The caseous centers enlarge and coalesce, and acid-fast bacilli are numerous, both in the areas of necrosis and within macrophages. In the final stages of AIDS, there is a pyohistiocytic response and myriad acid-fast bacilli.

Fungi Candidiasis. Candidiasis of the oropharynx, vulva, or vagina that is persistent, frequent, or poorly responsive to therapy increases in frequency starting in the middle stages of H1V infection. Neither oral nor vaginal candidiasis is highly predictive for H1V, because they are common complications of diabetes, antibiotic therapy, and other immunodeficient states. More than 90% of HIV-infected patients will develop one or more of the three forms oforopharyngeal candidiasis: erythematous (sometimes atrophic), pseudomembranous (thrush), and angular cheilitislS; some investigators also include hypertrophic (leukoplakia). 16 Candidal vuMtis and vaginitis are often the index condition in HIV-positive women, and present as mucosal erythema with a creamy white discharge. In H1V-infected patients, a switch from TH1to TH2-type cell-mediated immunity correlates with the progression to AIDS and may also increase susceptibility to mucosal candidiasis. 17 The diagnosis of candidiasis of any location is often made by the clinical appearance of the lesion; scrapings reveal necrotic debris and desquamated parakeratotic epithelium admixed with yeast and pseudohyphae. Biopsy specimens of the lesions at this stage of HIV infection may show fungal invasion of the superficial

epithelium, acanthosis, focal erosions, and a mild to moderate chronic inflammatory response. The fungal elements can be seen with PAS, GMS, or other fungal stains. Candidiasis of bronchi, trachea, lungs, or esophagus indicates severe immune suppression and, therefore, is an indicator of AIDS. Most cases are due to Candida albicans, but in patients receiving long-term antifungal therapy, other species may be isolated. Esophageal plaques and ulcers usually have a longitudinal orientation and are multifocal. Rare cases of inflammatory masses and gastric bezoars (fungus balls) are reported. Scrapings of the lesions reveal findings similar to those described previously. Biopsy specimens may show a more invasive form of the disease with mixed acute and chronic inflammation, erosions, and ulcers. Systemic candidiasis is rare in AIDS, probably because neutrophil function usually is adequate to limit the infection to the mucosal surface.

Coccidioidomycosis, disseminated or extrapulmonary. Coccidioidomycosis accounts for less than 1% of AIDSdefining conditions reported to the CDC, but is an increasingly important opportunistic mycosis in AIDS patients from endemic areas of the southwestern United States (especially Arizona), Mexico, and portions of Central and South America. The infection is acquired through inhalation of arthrospores; dissemination may be hematogenous or lymphatic. Thick-walled, nonbudding spherules, the diagnostic form ofCoccidioides immitis, are seen on hematoxylin-eosin-stained sections, are PAS- and GMS-positive, measure from 30 to 100/xm, and often contain endospores. The spherules stain red in Papanicolaou-stained cytologic preparations (Fig 2). The endospores measure 5 to 30/xm and may be seen free in the tissue. In non-AiDS patients, the spherules elicit granulomatous caseation necrosis, whereas endospores

Figure 2. C immitis. Note the red-staining spherules containing endospores. (Bronchial wash, Papanicolaou stain; original magnification x400).

Pathology of HIV

may cause suppuration. Poorly formed granulomas are the usual response in persons with AIDS. 6 Cryptococcosis, extrapulmonary. Cryptococcosis presents as subacute meningitis or meningoencephalitis in the majority of HIV-infected patents infected by C~yptococcus neoformans. The organisms are ubiquitous, and most cases are probably newly acquired rather than reactivation of latent infections. Lung is the primary site of infection and the second most commonly affected organ in active disease. With time, nearly all infections will disseminate, often as papular, waxy lesions of the skin resembling molluscum. In tissue (within macrophages or giant cells, or in the extracellular matrix), one sees pleomorphic yeast that varies in size from 2 to 20 /xm and has single or multiple narrow-necked buds. Elongated tube forms are seen in the immunocompromised host. Although typically encapsulated with positive mucicarmine (pink-red) and alcian blue or Movat (deep blue-green) staining, capsule-defective forms are reported. Fontana-Masson can stain the cell walls of capsule-deficient forms, but this is not specific for cryptococci.18 The host response varies from minimal (mucoid, cystic) to granulomatous.

Histoplasmosis, disseminated or extrapulmonary. Histoplasmosis, presenting as a disseminated or extrapulmonary infection with Histoplasma capsulatum, is a common complication of HIV/AIDS in the United States and most other areas of the world.6,19Reactivation and newly acquired infections with this dimorphic fungus have been documented in endemic areas. Fever and weight loss are the most common clinical manifestations. Respiratory symptoms and chest radiograph abnormalities are seen in only half of the patients with disseminated disease. Lymph nodes, liver, spleen, and bone marrow are most frequently involved. Fungemia is common and can be diagnosed by blood culture. The organisms also can be seen on peripheral blood or bone marrow aspirate smears. In fluids and tissue, round to oval yeast measuring 2 to 4 btm with single buds are the predominate form. Multiple organisms are within the cytoplasm of reticuloendothelial cells; shrinkage artifact produces a small halo around the nuclear body on hematoxTlin-eosin staining. The yeasts are PAS- and GMS-positive, variably acid-fast, and often occur in clusters. Short hyphae may be present in some infections. The histologic response varies from classic caseating granuloma in persons with intact cellular immunity to a mixed inflammatory infiltrate with massive infection in the severely immunocompromised patient. Pneumoeystosis, any site. Pneumocystis carinii, once thought to be a protozoan, is probably a eukaryotic, primitive, sporulating fungus with a unicellular mycelial

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state. 2° Pneumocystosis occurs throughout the world almost exclusively in persons with impaired cell mediated-immunity, and the major reservoir for infection is probably human lung. 21P carinii pneumonia was seen in up to 85% of patients with AIDS at some stage during their illness22; with the use of prophylaxis and combination antiretroviral therapy, the prevalence is decreasing. Patients infected with HIV most frequently develop P carinii pneumonia when their CD4 count is below 200 cells//xL and they are off prophylaxis.%,24 Most cases of P carinii pneumonia are likely to have arisen from reactivation of latent infection, although recurrent disease may be due to reacquisition. 6,21 Most patients present with fever, cough, dyspnea, and bilateral, diffuse interstitial infiltrates on chest radiograph. 25 Extrapulmonary P carinii is reported mainly in patients with overwhelming pulmonary infections. Disseminated infection has been reported in almost every organ system, including the gastrointestinal tract, lymph nodes, skin, ears, eyes, bone marrow, spleen, liver, kidneys, and thyroid gland. ExtrapulmonaIy lesions are similar to those seen in the lung. 21 The most common histologic pattern is an alveolitis with interstitial infiltration by lymphocytes and plasma cells and an eosinophilic intra-alveolar foamy ("honeycomb") exudate in which the organisms are usually found. The alveolar lining cells may be hyperplastic. Diffuse alveolar damage, progressive fibrosis, and destructive, cavitary, and noncavitary nodular and granulomatous lesions also have been described. 6 The granulomatous form may closely mimic tuberculosis. 26 Clusters of organisms are centered in areas of necrosis (Fig 3A). There may be focal calcium deposits. Methenamine silver preparations show oval or cup-shaped slivered cysts that measure 4 to 7/xm (Fig 3B). Single or paired comma-shaped thickenings of the cyst wall may be seen in the center or along the edge of the cyst. Intracystic bodies and trophozoites can be seen with Giemsa or Wright stains. P carinii pneumonia may arise or recur in patients with concurrent infections (Fig 4). Immunofluorescent stains are highly sensitive and have acceptable specificity, but their use may be of most value in situations in which suspicion ofP carinii infection has not been confirmed by a silver stain. 27

Viruses Cytomegaloviral disease, including retinitis with loss of vision (other than liver, spleen, or nodes). Infection with cytomegalovirus (CMV), a double-stranded DNA herpes virus, affects 40% or more of HIV-infected patients with CD4+ T-cell lymphocyte counts less than 100 cells//xL.28,29 With increased numbers of patients

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Figure 3. (A) Pcarinii: large necrotizing granuloma of the lung, (Hematoxylin-eosin stain; original magnification xl0.) (B) P carinii: cysts within the necrotic area. (GMS stain; original magnification x250.)

surviving with low CD4 T-cell counts, the prevalence and incidence of CMV-related disease have increased. Most AIDS patients have persistent CMV viremia, and CMV is a major cause of dysfunction in a variety of

organs. The AIDS-defining sites of involvement include the entire gastrointestinal and biliary tract, retina, lung, brain, and adrenal glands. Infection of the lung can cause focal or diffuse interstitial and hemorrhagic pneumonia. 3° The incidence of CMV retinitis ranges from 12% to 27% in histologic series. 31 Ocular lesions may result in visual loss. The host response varies from minimal to acute ulceration or hemorrhagic necrosis. Cytomegalovirus induces cytomegaly, forms inclusions, and exhibits a predilection for growth in endothelial and mesenchymal cells. Glandular and surface epithelium may be involved. Vasculitis with subsequent thrombosis and ischemia accounts for much of the significant pathology. Rarely, CMV may induce pseudotumors composed of granulation tissue and fibrosis with areas of acute and chronic inflammation) 2 Intussusception has been reported. Because CMV may be present as an incidental finding in sputum, urine, or other fluids, diagnosis depends on the histologic identification of characteristic viral inclusions usually identified on hematoxylin-eosin stains in tissues. The enlarged nucleus of an infected cell possesses an oval ground-glass inclusion with a peripheral halo, the so-called "owl-eye" intranuclear inclusion. Cytoplasmic inclusions are coarse and granular, are positive with GMS and PAS, and are occasionally mistaken for bradyzoites of Toxoplasma gondii. Atypical ground-glass inclusions may be present, and the diagnosis can then be confirmed by immunohistochemistry or in situ hybridization (Fig 5). Oralhairy leukoplcd~ia. Oral hairy leukoplakia may occur either early or late in the natural history of HIV disease, becoming more common as the disease

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Figure 4. P carinii and MAC in a pulmonary intra-alveolar "honeycomb" exudate. (GMS & Ziehl Neelsen stain; original magnification x330.)

Figure 5. Cytomegalovirus in gastric mucosa showing intranuclear and intracytoplasmic inclusions on the left (Hematoxylineosin stain; original magnification x250) and CMV immunohistochemical staining on the right (CMV immunostain original magnification x250).

Pathology of HIV

progresses. Oral hairy leukoplakia presents clinically as raised, irregular, or corrugated white lesions usually on the lateral border or dorsal surface of the tongue. Histologic features include parakeratotic hyperkeratosis with elongation of the rete ridges. Vacuolated cells of the malpighian layer, beneath the parakeratotic layer, are similar in appearance to koilocytes. Histologic examination reveals koilocytes within all layers of the epithelium, a variable degree of acanthosis, parakeratosis, and fine hair-like projections of keratin. The presence of Epstein-Barr virus on electron microscopy, immunohistochemistry, in situ hybridization, or polymerase chain reaction is required by some investigators to make a definitive diagnosis.25 Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome and~or herpes simplex (chronic ulcers > 1 month duration, or bronchitis, pneumonitis, or esophagitis). There is decreased specific immune response to viral infections (especially DNA viruses) in HIV infection because of defective cytotoxic function of CDS+ lymphocytes. Mucocutaneous herpes simplex (human herpesvirus 1 and 2) and herpes zoster are early and recurrent causes of morbidity. The ulcers in severely immunocompromised patients are often large and multiple, and frequently recur and persist33 Herpes zoster may present in multiple dermatomes concurrently or sequentially. Presumptive diagnosis can be made from cytologic scraping of the lesion or biopsies of the active border. On biopsy specimens there is acantholysis, erosions, and/or ulceration, as well as a variable degree of acute and chronic inflammation. Cells infected by herpes simplex virus are often multinucleated with ground-glass or eosinophilic (Cowdry type A) intranuclear inclusions. If necessary, viral culture, in situ hybridization, or immunohistochemistry can be used to differentiate herpes simplex from zoster. Progressive multifocal leukoencephalopathy. Progressive multifocal leukoencephalopathy is a demyelinating disease of the central nervous system caused by a papovavirus (JC virus) infection ofoligodendrocytes that occurs in 4% to 5% of AIDS patients. The patient develops focal neurologic defects, such as hemiparesis, visual defects, ataxia, or cognitive difficulties. In most HIV-infected patients, progressive multifocal leukoencephatopathy progresses to death within an average of 4 months. Computed tomography reveals nonenhancing, hypodense lesions, often in the parieto-occipital lobes. Definitive diagnosis requires biopsy and in situ hybridization or electron microscopy of the virus. On gross examination of the brain, areas of demyelination are

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easily identified, but biopsy specimens from the brain may be too small or fragmented to identify these changes. Sections reveal demyelination, enlarged hyperchromatic oligodendroglial nuclei, and enlarged bizarre astrocytes.34

Protozoa

Cryptosporidiosis, intestinal, chronic (>1 month's duration). Cryptosporidiosis is a zoonotic coccidian infection of the gastrointestinal and, rarely, respiratory tracts. Cryptosporidial oocysts are frequently present in surveys of surface water in the United States and South America and may contaminate municipal water supplies unless proper filtration is used. Infections in the immunocompetent host are often self-limited, but immunocompromised patients develop chronic diarrhea. To be an AIDS-defining illness, cryptosporidiosis must have a duration of more than 1 month; such chronic infections usually occur in persons with CD4+ T-cell counts less than 200 cells//xL. It is estimated that cryptosporidia are found in 10% to 20% of A1DS patients with chronic diarrhea. The infection may spread to the biliary tract, causing cholecystitis, sclerosing cholangitis, hepatitis, or pancreatitis. Sinonasal and puhnonary cases also have been reported. Diagnosis can be made on Kinyoun carbolfuchsin staining of oocysts in stools. Hematoxylin-eosin staining of mucosal biopsy specimens indicate that the oocysts are basophilic, measure from 4 to 6/xm, and are located along the brush border of the epithelium in an intracellular but extracytoplasmic location. The brush border may be altered, and there is an associated chronic inflammatory infiltrate of the lamina propria. Mucosal damage includes villous atrophy and inflammatory cell infiltration through the wall extending to the lamina propria. The acid-fastness present on direct smears is usually not present in fixed tissue.35 Isosporiasis, intestinal, chronic (>I month's duration). Isosporiasis is a coccidian parasite that occasionally causes chronic, AIDS-defining diarrhea. Extraintestinal dissemination has been rarely reported. Isospora belli occurs more frequently in the developing world than in the United States. The primary site of infection is the enterocytes of the small intestine. Small numbers of sporozoites, schizonts, and merozoites are seen in subnuclear vacuoles, free in the lumen, in the interstitium, a n d in mucosal lymphoid tissue. Villi are somewhat flattened or totally atrophic. The organisms are visible on hematoxylin-eosin-stained sections and vary in size from 3 to 15/xm spherical bodies to 12 × 30/xm ellipsoid

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forms. Oocysts can be identified by acid-fast stains of stool.35 Toxoplasmosis of the brain. Toxoplasmosis of the brain, one of the most common neurologic complications of AIDS, affects 5% to 25% of patients with CD4+ T-cell counts of under 100 cells//xL. T gondii is an obligate intracellular coccidian that is transmitted to humans as infective oocysts from felines or by ingesting the undercooked meat of infected animals. It is the most common parasitic infection in the United States, but rarely causes significant disease in immunocompetent individuals. Reactivation, rather than newly acquired infections, is thought to be the major source of disease. Clinical symptoms often have an acute onset, with both diffuse and focal neurologic defects. Computed tomograplay reveals multiple ring-enhancing or mass lesions in the basal ganglia and cortex. Histologically, vasculitis with surrounding hemorrhage and necrosis should suggest a diagnosis of Toxoplasma encephalitis. Definitive diagnosis requires visualization for the characteristic tachyzoites or cysts in tissue; hematoxylin-eosin is usually adequate. In fixed tissue, the cysts measure 5 to 100/xm and contain many bradyzoites. Tachyzoites are 2 to 6/zm crescentic forms that may be seen in groups, intracellularly or extracellularly, and may appear rounded in f~xed tissue. Immunohistochemical stains may increase the sensitivity, but there is wide variation in the specificity of these antibodies. 36 Nonindicator Conditions

Bacteria Botryomycosis.

Botryomycosis is an infectious process caused by nonfilamentous bacteria in the order Eubacteriales that stimulates grain formation. Known causative agents include Staphylococcus aureus (most common), Streptococcus sp, Pseudomonas aeruginosa, Escherichia coli, and Proteus vulgaris. The lesions are usually localized in the skin and soft tissue, but may occur in any organ or tissue. Visceral involvement is rare and usually results from hematogenous seeding. Lung and lymph node are the most common viscera involved. Grains are present in suppurative loci that are frequently accompanied by a granulomatous reactionY Organisms are identified on tissue gram stain (Fig 6). In severely immunocompromised patients, organisms are also found outside the grains, a feature not seen in most grain-forming infections in immunocompetent hosts. Rhodocorcus equi. R equi is an aerobic, intracellular, gram-positive, partially acid-fast coccobacillus that was

Figure 6. Botryomycosis, lymph node. A grain containing gram-positive staphylococci is seen with tissue gram stain. Organisms are also seen in phagocytic cells outside the grain. (Brown-Hopps stain; original magnification x66.)

originally identified in horses. The organism has a worldwide distribution and is found in soil.% R equi infection in humans is uncommon and predominantly affects immunocompromised patients. A history of contact with farm animals is often absent. In HIV-infected individuals, this infection tends to occur when CD4 counts decrease to less than 50 cells//~L.39,4° The most frequent presentation is pneumonia; patients may develop cavitation, pleuritis, pleural empyema, and histiocytic pseudotumors of the bronchi. 39< Although sensitive to antibiotics, R equi infections frequently recur and often are fatal. 4° In HIV-infected patients, the histologic features of pulmonary malakoplakia, which were rarely reported before the HIV era, are often present. Sheets of macrophages with foamy eosinophilic cytoplasm (von Hansemann cells) stain with PAS and GMS, and contain lamellated calcified cytoplasmic Michaelis-Gutmann bodies.42Michaelis-Gutmann bodies may be scant. Focal necrosis and collections of neutrophils and many intracellular and extracellular gram-positive coccobacilli can be seen (Fig 7). The organism may be mistaken for normal flora, a contaminant, or a mycobacterium resulting from its partial acid-fast property seen particularly in smears and culture. 39,43R equi is most frequently isolated from blood culture, but can be cultured from sputum or bronchoalveolar lavage specimens. 4° Intestinal spirochetosis. Intestinal spirochetosis is considered to be an infestation of intestinal epithelium by spirochetes and, as such, requires a biopsy for diagnosis. The incidence in European patients undergoing lower gastrointestinal biopsy varies from 2.5% to 16.5%. In homosexual men, intestinal spirochetosis has

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Figure 7. (A) R equbassociated pulmonary malakoplakia. Sheets of foamy macrophages and an intracytoplasmic Michaelis-Gutmann body (arrow) are present. (Hematoxylin-eosin stain; original magnification x250.) (B) Pulmonary R equi infection showing multiple intracytoplasmic and extracytoplasmic gram-positive coryneform bacteria. (Brown-Brenn stain; original magnification x250.)

been seen in 30% of rectal or colonic biopsy specimens from both H1V-infected and noninfected patients. 44 A heterogeneous group of spirochetes is probably involved, including Brachyspora aalborgii and a Treponema organism formerly identified as Spirochaeta eurygyrata, seen mainly in Middle Eastern and African populations. Intestinal spirochetosis is associated with many conditions with unrelated etiologies and may arise secondarily to chronic stasis. In HIV-infected patients, chronic diarrhea, not attributable to other infectious causes, improved with clearance of spirochetes after antibiotic treatment. 44 Histologically a 2- to 3-/xm basophilic fringe on the luminal aspect of enterocytes in hematoxyqin-eosinstained sections extends a short distance into the neck of crypts.44There may be a mild inflammatory response in the lamina propria, but usually crypt architecture is not altered. The organisms are well demonstrated by silver impregnation techniques, such as the Warthin-Starry stain (Fig 8).

inclusions. Immunohistochemical stains are confirmatory~ (Fig 9). Parvovirus B19. Parvovirus B19 is associated with chronic infection and anemia in the immunocompromised host. 45Because these patients are often unable to produce neutralizing antibodies, they do not clear the infection.46 Normochromic normocytic anemia and bypercellular marrow are the most consistent findings. Immunoglobulin therapy may eliminate or ameliorate the infection.45,46Parvovirus infection is more likely to cause severe anemia than other causes of chronic anemias in AIDS (eg, zidovudine, trimethoprim-sulfamethoxazole, or HIV-induced marrow dysplasia). Bone

Viruses

Adenovirus. Adenovirus may be an infrequent, yet significant, cause of lung, liver, and gastrointestinal disease in persons with H1V infection. Immunosuppressed patients may develop extensive, sometimes necrotizing, bronchitis, bronchiolitis, or enteritis. Fatal hepatic necrosis also has been described. The virus may be identified in tissue by the presence of characteristic smudge cells containing nuclei filled with basophilic material that obscures the nuclear membrane. Cells may contain amphophilic or eosinophilic intranuclear

Figure 8. (Left) Intestinal spirochetosis, colon. A gray haze replaces the normally pink luminal brush border. (Hematoxylineosin stain; original magnification x250.) (Right) Silver impregnation blackens the spirochetes coating the luminal surfaces of enterocytes. (Warthin-Starry stain; original magnification x250.)

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stains to identify the division process should allow the observer to distinguish the two.4s Parasites

Amebiasis. Acanthamoeba sp (eg, Acanthamoeba castellani, Acanthamoeba culbertsoni,Acanthamoeba rhysodes,Acanthamoeba polyphagia) and leptomyxids (eg, Balamuthia mandrillaris) are free-living amebae found in soil and water

Figure 9. (Left) Adenovirus, lung. A large basophilic intranuclear inclusion is present. (Hematoxylin-eosin stain; original magnification x250.) (Right) Positive immunohistochemical staining for adenovirus. (Adenovirus immunostain; original magnification x250.)

marrow aspirates and biopsy specimens show erythroid hypoplasia (Myeloid:Erythroid ratios > 5:1), decreased or arrested erythroid maturation, numerous giant pronormoblasts, and increased iron (personal observations). There may be margination of the nuclear chromatin or intranuclear halos, the so-called "lantern cells" (Fig 10). In paraffin-embedded, formalin-fixed material, large pink to lilac intranuclear inclusions may be seen. In contrast to herpes simplex and CMV, the inclusions are seen only in erythroid cells. 47 Immunohistochemistry is often required to confirm the diagnosis.

throughout the world. Species able to tolerate temperatures above 37°C are most likely to cause human disease. Both cyst and trophozoite stages exist in nature and are identifiable in tissue sections. Transmission may occur via respiratory mucosa or contaminated skin lesions; spread is hematogenous. Aeanthamoeba sp and Balamuthia sp infections are increased in contact lens wearers and in the immunocompromised host. Acanthamoebiasis classically presents as granulomatous meningoencephalitis and keratoconjuctivitis, but chronic skin or mucosal lesions occur before or coincidental with

Fungi Penicillium marneffei. P marneffei is a dimorphic fungus endemic to Southeast Asia. Before the AIDS epidemic, penicilliosis was a rare infection of immunocompromised hosts. Both the incidence and prevalence have increased dramatically in the last decade. It is currently the third most common AIDS-related opportunistic infection in northern Thailand (after tuberculosis and cryptococeosis). Cutaneous and subcutaneous abscesses and disseminated infection involving the mononuclear phagocytic system are the usual presentations. As with other intracellular infections, the host response varies with the integrity of the immune system from granulomatous to suppurative to anergic. Within macrophages, these nonbudding yeast are round to oval and measure 2 to 6/zm, but elongated forms may reach 12/xm in the extracellular location. Reproduction is by schism of elongate forms; the central transverse septum stains well with GMS and is thicker than the cell wall. Differential diagnosis includes H capsulatum. Fungal

Figure 10. (A) Parvovirus B19, bone marrow. A giant pronormoblast with margination of nuclear chromatin ("lantern cell"). (Hematoxylin-eosin stain; original magnification x250.) (B) Parvovirus B19, bone marrow. Immunohistochemical stain for hemoglobin highlighting giant pronormoblasts. (Hemoglobin immunostain; original magnification x 100.)

Pathology of HIV

central nervous system disease in a significant percentage of cases. Skin and mucosal lesions begin as papules and nodules and progress to ulcers. The ulcers a r e poorly demarcated, have elevated borders, and eventually heal. Cerebral abscesses have been reported in persons with AIDS. Trophozoites in tissue are 8 to 20 /xm, and the nucleus has a large central ("bull's eye") karyosome (Fig 11). B mandrillaris, a member of the order Leptomyxida, causes granulomatous encephalitis, especially in immunocompromised persons, and is found in other tissues such as the kidney and adrenal. Trophozoites are 15 to 35/.~m. Both Acanthamoeba sp and B mandrillaris may be present in tissue as cysts that have a thick, irregular ectocyst; the endocyst shrinks on fixation, leaving a space. The cyst wall does not stain well with hematoxylin-eosin, but silver impregnation (GMS, Warthin-Starry) and PAS may accentuate this structure. Balamuthia sp may have double nuclei; the karyosome is less prominent than those ofAcanthamoeba sp. The species usually cannot be determined by morphologic features; immunohistochemical stains, electron microscopy, or cultures are required. 49These organisms a r e distinguishable from another agent of amebic meningoencephalitis, Naegleriafowleri, which is smaller (7 to 15 /xm) and does not encyst in tissue9 Histologically, there is granulomatous inflammation with a marked cellular response consisting of macrophages, lymphocytes, and plasma cells. A neutrophilic infiltrate may be present. Skin lesions in the immunocompromised host may show an altered response with lack of giant cells and only poorly formed granulomas; leukocytoclastic vasculitis has been reported. 5° Variable numbers of organisms are found throughout the lesion and may surround dermal vessels (Fig 11).

Figure 11. Acanthamebic sinusitis. Trophozoite in an area of necrosis. Note the "bull's eye" karyosome. (Hematoxylin-eosin stain; original magnification x250.)

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Leishmaniasis. Leishmaniasis is caused by various species of the dimorphic protozoa Leishmania. The spectrum of diseases caused by these organisms has been grouped into visceral, mucocutaneous, and cutaneous leishmaniasis. Leishmaniasis is prevalent throughout Asia, Africa, the Americas, and the Mediterranean, with up to 1.5 million new cases diagnosed each year.51 Approximately one third of the world's HIV-infected population lives in zones endemic for leishmaniasis. The largest number of co-infections (>750) have been reported from southern Europe (Spain, Italy, France, and Portugal); however, Brazil, Ethiopia, Kenya, and Tunisia have also reported endemic cases. Most have been reported in intravenous drug users, but this is the most common HIV risk factor in the populations studied. 52In the United States, most cases are acquired from endemic areas, although Leishrnania mexicana has been reported in cutaneous infections from Texas. Both reactivation and new infections occur. Visceral dissemination is common, even with the typically cutaneous species. Treatment failure and relapse are also common. Death during the first episode of visceral leishmaniasis has been reported in up to 19% of patients) 2 The diagnosis of leishmaniasis ultimately depends on identification of the organism from tissue, by direct smear or by culture. 53 Common to all leishmanial lesions are infiltrates of large histiocytes and varying numbers of organisms. Organisms are more numerous in early lesions and those arising in the immunocomproraised host. Amastigote forms (Leishman-Donovan bodies) are generally found intracellularly and are 1 to 3/xm in diameter, round to oval, nonencapsulated, and have a distinct cytoplasmic membrane. In routine hematoxylineosin-stained sections, they have a round, basophilic nucleus approximately 1/xm in diameter and a small basophilic rod-like kinetoplast. Various admixtures of lymphocytes, neutrophils, and plasma cells are also present. Lack of budding and presence of the kinetoplast a r e features used to distinguish Leishmania sp from Histoplasma sp and Toxoplasma sp. In addition, fungal stains can help identifyHcapsulatum. Microsporidiosis. Although microsporidiosis is not yet an indicator condition of the CDC surveillance definition, it is found in ->20% of AIDS patients with diarrhea. Microsporidia are tiny obligate unicellular zoonotic parasites that contain ribosomes, lack mitoehondria, and have a coiled polar tube injection apparatus. Microsporidia have great reproductive potential, multiplying within cells and easily spreading fi~om cell to cell. The most common species identified in humans are Enterocytozoon bieneusi, Encephalitozoon hellum, and Encepha-

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Figure 12. E bieneusi spores are seen in the supranuclear area of the enterocytes of small intestine as small bluish bodies. (Brown & Hopps stain; original magnification x330.) litozoon (Septata) intestinalis. In the immunocompromised host, infections by E hellum and E intestinalis are often disseminated, but, unlike E bieneusi, respond to treatment with albendazole. E bieneu,i is usually limited to the intestinal and respiratory tract epithelia. 54 The organisms are easily overlooked in tissue biopsy specimens because of their small size (1 to 3/xm). Light microscopic tissue diagnosis can be made with hematoxylin-eosin staining, but the organisms are more easily identified with tissue gram stain. Some species have a gram-positive equatorial belt or a PAS-positive polar cap; others are birefringent (especiallywith gram stains). Spores of E bieneu~i are bluish, refractile, and found in supranuclear vacuoles of the glandular epithelium (usually in enterocytes, but also in the biliary, respiratory, and urinary tracts and in the pancreas) (Fig 12). E intestinalis and E hellum infect both epithelial and reticuloendothelial cells. Spores ofE intestinal# may be present within a multiloculated parasitophorous vacuole. The histologic changes vary from a mild nonspecific inflammation to ulceration and acute suppurative inflammation. We have seen several cases of sinonasal polyps and one case each with fibrotic, punched-out lesions of the scalp and mandible. Determination of the species requires electron microscopy or immunodiagnosis. Strongyloidiasis. Strongyloidiasis is considered one of the "missing infections" in AIDS. In areas with a high prevalence of Strongyloides stercoralis infection, such as central Africa, the HIV-infected population does not appear to have a higher incidence of dual infection or a greater frequency of hyperinfection.55Patients with H1V who were not exposed to S stercoralis before the acquisition of H1V may lack protective immunity and be at higher risk for dissemination. 56Reactivation and disseminated infection can occur in association with the use of

immunosuppressive drugs. 57Infective larvae ofS stercoral/s from soil enter the skin, invade the lymphatics, and travel through the venous system to the right side of the heart and lung. They migrate to the trachea, are swallowed, and reach the duodenum and proximal jejunum, where they are able to multiply. Eggs develop into larvae that may be expelled with feces or metamorphose into infective forms that can invade the gastrointestinal wall. Migrating filariform larvae may appear in any organ, most frequently the lung, where they can cause hemorrhage. Parasites may be seen in bronchoalveolar lavage in either disseminated or nondisseminated infections. In the gut, parasites are most frequently present in the duodenum and proximal jejunum.

Conclusion Infections are the most common complication of HIV infection, and most are amenable to treatment. Culture remains, in most instances, the "gold standard" for the identification of microorganisms. However, in some instances, especially in immunocompromised hosts, histologic or cytologic identification is relied on (eg, P carinii pneumonia). The identification of etiologic agents in tissue is often more rapid than in culture, and multiple co-infections may be identified simultaneously using appropriate special stains. The advent of molecular diagnostic testing for infectious microorganisms has added to our abilities to detect many organisms with greater speed and sensitivity, but the identification of causative agents within lesions remains an important role for the pathologist.

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