Context.Penicillium marneffei, an opportunistic fungus, is endemic in Southeast Asia, especially in human immunodeficiency virus–infected individuals living in northern Thailand.

Objective.—We present the results of a clinicopathologic study of hepatic penicilliosis among human immunodeficiency virus/acquired immunodeficiency syndrome patients.

Design.—A search of liver biopsies in one institution from 1998 to 1999 identified 30 cases of penicilliosis.

Results.—Histologically, hepatic lesions could be classified into 1 of 3 patterns: diffuse, granulomatous, and mixed. The diffuse pattern showed a diffuse infiltration of foamy macrophages that contained numerous P marneffei. The granulomatous pattern showed a formation of multiple granulomata with various degrees of inflammatory cell infiltration. The mixed pattern showed features intermediate between the diffuse and granulomatous patterns. Liver function tests of the 3 pathologic pattern groups were evaluated, but there were no statistically significant differences in aspartate aminotransferase, alanine aminotransferase, or alkaline phosphatase levels among the various histologic groups.

Conclusion.—To our knowledge, this is the largest series to date that documents the liver pathology that results from this pathogen. We hypothesize that the histologic patterns seen on biopsy reflect the level of the host's immunity. Hence, in addition to a diagnosis of penicilliosis, a liver biopsy may also provide an assessment of the host's immune status, whereas liver function tests do not.

P;thenicillium marneffei is an endemic fungus in Southeast Asia that causes deep-seated infection in humans and rodents. Penicillium marneffei was first reported in Vietnam in 1956, following its isolation from the liver of a bamboo rat (Rhizomys sinensis) that died of disseminated infection.1 The first case of a natural human infection was described in 1973 in an American with Hodgkin disease who had a travel history to Southeast Asia.2 More recently, P marneffei infection has become one of the major opportunistic infections among human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) patients in Southeast Asia, particularly in northern Thailand.3–8 

In most cases, the fungus invades the reticuloendothelial system and causes a deep-seated infection that can be focal or disseminated.3,4,9–12 Hepatic involvement of P marneffei is frequently observed among HIV/AIDS patients, but there are only isolated reports as parts of larger studies or as single case reports, and these are often from postmortem material only.13–19 To our knowledge, no report specifically details the clinicopathologic features of hepatic penicilliosis. In this study, we report the biopsy results of 30 HIV/AIDS patients from northern Thailand who had hepatic involvement by P marneffei. We found that a liver biopsy is reliable in making a diagnosis of hepatic penicilliosis and that there are different patterns of pathologic damage that, we hypothesize, reflect the level of host immunity.

Patient Population

Thirty HIV-infected patients with disseminated P marneffei infection, all of whom were admitted to Chiang Mai University Hospital from 1998 to 1999, were included in this study. Diagnosis of HIV infection was made if the patient's serum was repeatedly reactive by both enzyme-linked immunosorbent assay methods (Enzymun-Test, Anti-HIV 1 + 2, Boehringer Mannheim Gmbh Diagnostica, Germany) and particle-agglutination tests (Serodia-HIV, Fujirebio Inc, Tokyo, Japan). These methodologies do not distinguish HIV-1 and HIV-2. For that reason, throughout this article, the less specific term of HIV is used. The diagnosis of P marneffei was made by the isolation of organisms from patient blood.

Liver Function Studies

All patients underwent liver function tests, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin. Statistical analysis was performed using the Kruskal-Wallis test. P values less than .05 were considered significant.

Histopathology

The liver biopsy specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections were cut at 3 to 4 μm and stained with hematoxylin-eosin, periodic acid–Schiff, Azan-Mallory, Grocott methenamine silver, and Ziehl-Neelsen.

Histopathologic Results

Penicillium marneffei was identified by Grocott methenamine silver staining as a round-to-oval yeastlike organism measuring 2 to 7 μm with central septa and a lack of budding forms (Figure 1). These features ruled out the organism as being Cryptococcus and Histoplasma. None of the biopsies of these patients showed multiple infections. Lesions in the liver biopsy specimens were classified into 1 of 3 patterns. A “diffuse” pattern was seen in 10 cases with widespread infiltration of foamy macrophages, mainly in sinusoidal spaces and focally in portal tracts, without a significant infiltration of lymphocytes, neutrophils, or other inflammatory cells. Liver cell cords were compressed and/or atrophic. The macrophages contained numerous P marneffei (Figure 2). A “granulomatous” pattern was present in 5 cases with formation of multiple granulomata in the liver parenchyma and portal tracts with mild-to-moderate inflammatory cell infiltration (Figure 3). Small numbers of P marneffei were identified in both intracellular and extracellular locations. A “mixed” pattern was seen in 15 cases with features intermediate between the diffuse and granulomatous patterns (Figure 4).

Figure 1.

Penicillium marneffei is identified as a round-to-oval yeastlike organism measuring 2 to 7 μm with central septa (arrows) and a lack of budding forms (Grocott methenamine silver, original magnification ×400). Figure 2. Diffuse pattern with widespread infiltration of foamy macrophages in sinusoidal spaces without significant infiltration of other inflammatory cells (hematoxylin-eosin, original magnification ×100). The infiltration is apparent on periodic acid–Schiff (PAS) staining, which also demonstrates numerous Penicillium marneffei within macrophages (inset; PAS, original magnification ×400). Figure 3. Granulomatous pattern with formation of multiple granulomata and mild inflammatory cell infiltration in the liver parenchyma (hematoxylin-eosin, original magnification ×100). Short arrows indicate small number of extracellular Penicillium marneffei (inset; periodic acid–Schiff, original magnification ×400). Figure 4. Mixed pattern with features intermediate between the diffuse and granulomatous patterns. Long arrows mark granulomata (hematoxylin-eosin, original magnification ×100). Short arrow indicates macrophages that contain Penicillium marneffei (inset; periodic acid–Schiff, original magnification ×400)

Figure 1.

Penicillium marneffei is identified as a round-to-oval yeastlike organism measuring 2 to 7 μm with central septa (arrows) and a lack of budding forms (Grocott methenamine silver, original magnification ×400). Figure 2. Diffuse pattern with widespread infiltration of foamy macrophages in sinusoidal spaces without significant infiltration of other inflammatory cells (hematoxylin-eosin, original magnification ×100). The infiltration is apparent on periodic acid–Schiff (PAS) staining, which also demonstrates numerous Penicillium marneffei within macrophages (inset; PAS, original magnification ×400). Figure 3. Granulomatous pattern with formation of multiple granulomata and mild inflammatory cell infiltration in the liver parenchyma (hematoxylin-eosin, original magnification ×100). Short arrows indicate small number of extracellular Penicillium marneffei (inset; periodic acid–Schiff, original magnification ×400). Figure 4. Mixed pattern with features intermediate between the diffuse and granulomatous patterns. Long arrows mark granulomata (hematoxylin-eosin, original magnification ×100). Short arrow indicates macrophages that contain Penicillium marneffei (inset; periodic acid–Schiff, original magnification ×400)

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Liver Function Test Results

The results of liver function tests for the 30 cases of hepatic P marneffei are presented as follows: the highest elevation of AST was found in the diffuse pattern (mean = 242 U/L, SD = 262), followed by the mixed pattern (mean = 229 U/L, SD = 189) and the granulomatous pattern (mean = 103 U/L, SD = 45). The mixed pattern showed the highest level of ALT (mean = 64 U/L, SD = 55), followed by the diffuse pattern (mean = 90 U/L, SD = 59) and the granulomatous pattern (mean = 58 U/L, SD = 56). The highest ALP level was observed in the mixed pattern (mean = 370 U/L, SD = 376), followed by the granulomatous pattern (mean = 502 U/L, SD = 330) and the diffuse pattern (mean = 482 U/L, SD = 257). There was no statistical difference in AST, ALT, and ALP values among the 3 different pathologic patterns of hepatic penicilliosis using the Kruskal-Wallis test (P = .24, .25, and .38, respectively). The level of total bilirubin in the mixed pattern was higher than in the diffuse and granulomatous patterns, with mean values of 1.89, 0.81, and 0.80 mg/dL (32.3, 13.9, and 13.7 μmol/L), respectively (P = .02).

Penicillium marneffei belongs to the genus Penicillium and shows a characteristic thermal dimorphism. Within cells such as histiocytes, P marneffei is a round-to-oval yeastlike organism that measures 2 to 7 μm in diameter. Extracellular P marneffei is frequently more elongated and can measure up to 15 μm in length. For diagnostic purposes, the dividing cells of P marneffei contain an obvious central septum, and no budding forms are seen.20 These 2 characteristic features allow P marneffei to be distinguished from other similar-appearing fungal organisms that might occur in liver biopsies in a similar clinical setting, such as Histoplasma and Cryptococcus. In our review of liver biopsies, there were 33 cases that were histologically diagnosed as fungal infection, 30 of which were penicilliosis (forming the basis of this study), plus 2 cases of cryptococcosis and 1 of mucormycosis.

The fungus was first reported in Vietnam in 1956, following its isolation from the liver of an infected bamboo rat (Rhizomys sinensis).1 The first case of a natural human infection was described in 1973 in an American with Hodgkin disease who had traveled to Southeast Asia.2 Cases continue to be described in seemingly healthy individuals who travel to or live in the endemic regions located in Thailand, China, Hong Kong, Vietnam, Indonesia, Singapore, and Burma.21 In 1988, the first case of penicilliosis in an HIV-infected tourist to Southeast Asia was reported.22 Since that time, most cases have occurred among AIDS patients living in the Chiang Mai area of northern Thailand.3–8 ,Penicillium marneffei infection has now became the third most common opportunistic infection in AIDS patients in Chiang Mai, following extrapulmonary tuberculosis and cryptococcal meningitis.4 The number of infected patients reached more than 1300 by 1995.23 To put this number in perspective, the first cases of AIDS in Thailand occurred in 1984, and by 1995, approximately 800 000 Thais were infected with HIV; by the year 2000, 1 million Thais had been infected. More than 95% of the cases are recombinant subtype A/E, and the rest of the cases are subtype B′.8 Both subtypes experience similar opportunistic infections, levels of immunosuppression, and mortality rates.24 

Most cases of penicilliosis are disseminated in nature,9 with fever, generalized lymphadenopathy, hepatosplenomegaly, mucosal ulcers, and skin lesions.3,4,10–12,16,25 Hepatic involvement is frequent, but to date, to our knowledge, there is no report detailing the histologic or clinical features of hepatic penicilliosis. There is isolated mention of liver dysfunction among HIV patients with P marneffei infection, with mild-to-moderate elevations of serum AST, ALT, ALP, and total bilirubin levels.26,27 Similarly, there are surveys of HIV-infected patients who have undergone liver biopsies that have found that 3% to 9% of these patients were diagnosed with penicilliosis.15,18 These are the largest series to date, with each one including 4 patients. There has been no attempt to correlate liver function and pathology findings in hepatic penicilliosis.

To our knowledge, our study of 30 cases of hepatic penicilliosis is now the largest series available. We found that hepatic lesions of P marneffei infection could be classified into 1 of 3 histologic patterns: diffuse, granulomatous, and mixed. These patterns occurred in approximately equal proportions, although slightly more cases were in the mixed pattern than in the other groups. The diffuse pattern reflected a pattern of macrophage infiltration of the liver sinusoids and parenchyma, without discrete granuloma formation and a general paucity of other types of inflammatory cells. The granulomatous pattern reflected the presence of well-formed granulomata within the liver parenchyma, in various sizes and numbers, accompanied by other types of inflammatory cells. Biopsies with the mixed pattern showed features intermediate between the other 2 more distinct patterns.

We hypothesize that these different patterns of hepatic involvement reflect the level of host immunity (the degree to which cell-mediated immune function has been suppressed). A similar phenomenon is seen with other infections, such as leprosy, leishmaniasis, and fungal diseases including cryptococcosis and sporotrichosis. As an example, Mycobacterium leprae infection displays 2 principal subtypes, tuberculoid and lepromatous, which reflect host immunologic response.23 A borderline type develops in patients whose resistance lies between the 2 major types. The tuberculoid type occurs in the setting of a strong immunologic response with epithelioid granuloma formation containing few bacilli and a lymphocyte reaction. On the contrary, cellular resistance against organisms is poor in the lepromatous subtype, which is characterized by a diffuse infiltration of foamy histiocytes and a large number of bacilli. Borderline leprosy shows an intermediate appearance between the tuberculoid and lepromatous types.

In humans, nonhepatic tissues infected with P marneffei can show one of three patterns: suppuration and granuloma formation and anergic with necrosis. The latter is more commonly seen in immunocompromised patients.9 Experimental work with P marneffei has shown that mice develop infection following exposure by either a respiratory or parenteral route, with hepatic involvement.28,29 Immunocompetent mice developed a granulomatous response to the organism with an eventual elimination of the pathogen. Congenitally immunodeficient mice initially formed granulomata, but they then developed progressive systemic disease. At this stage, there was a replacement of the liver parenchyma by free yeastlike cells and a proliferation of macrophages containing yeastlike organisms. Using athymic mice, cell-mediated immunity was shown to play a central role in the host defense against the organism.29 

On the basis of this work, we propose that the granulomatous pattern seen in patients reflects a more intact level of cell-mediated immune function and that the diffuse pattern reflects a more depleted level, with the mixed pattern somewhere in between. It would be of value to correlate these patterns with viral load and CD4 counts for individual patients; however, these data were not available. There are only a few pathology reports of hepatic penicilliosis in the literature with enough detail to enable us to categorize the findings into 1 of our 3 patterns. Cases with the granulomatous pattern were seen in HIV-positive patients being investigated for abnormal liver function tests18 and in one HIV-negative patient with an autoimmune disease.19 Cases with the diffuse pattern were from postmortem material of patients succumbing to HIV infection.9,13,14,16 

In our study, we also attempted to correlate liver function testing with pathology. The diffuse pattern tended to show injury involving hepatocytes to a greater extent than bile ducts. The highest mean value for AST occurred in this group as well as the lowest mean value for ALP. The granulomatous pattern appeared to be associated with the least severe liver dysfunction. This group had the lowest mean values for AST, ALT, and total bilirubin. The mixed pattern was associated with the most severe liver disease. In this group, the highest mean values for ALT, ALP, and total bilirubin were observed, and the mean value for AST (mean = 229 U/L) was just slightly less than the highest value (mean = 242 U/L), which was observed in the diffuse-pattern group. Elevations of ALP were seen in all 3 groups, which implies that damage to bile ducts occurs with all patterns of P marneffei infection but is greatest in the mixed-pattern group. However, none of these liver function tests had significant predictive values with respect to the pattern of liver involvement by penicilliosis.

In conclusion, a spectrum of pathologic damage to the liver is observed in cases of hepatic penicilliosis. This damage can be grouped into 1 of 3 morphologic patterns, but there is poor correlation with the degree of liver dysfunction. We propose that these patterns reflect the degree of immunocompetence in the patient population we were studying, all of whom were infected with HIV. We do not know if the same patterns and correlations apply to healthy individuals infected with P marneffei, since we did not have such individuals in our study group. Nevertheless, within our patient group, a liver biopsy appears to be useful not only for the diagnosis of P marneffei, but perhaps also for predicting immune status.

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Author notes

Reprints: Amnat Yousukh, MD, Department of Pathology, Faculty of Medicine, Chiang Mai University, 110 Intavaroros Rd, Chiang Mai 50200, Thailand ([email protected])