_
REPORT
IMMUNOHISTOCHEMICAL IDENTIFICATION OE PENICILLIUM MARNEFFEI BY MONOCLONAL ANTIBODY JORGE ARRESE ESTRADA, M.D., DIRK STYNEN, PH.D., JAN VAN CUTSEM, PH.D.,
CLAUDINE PIERARD-ERANCHIMONT, M.D., AND GERALD E. PIERARD, M.D.
Abstract
Materials and Methods
We present an experimental study on the immunohistochemical identification of Penicillium marneffei in paraffinembedded, formalin-fixed tissue. The monoclonal antibody EB-A1 detects a specific galactomannan that appears to have at least one epitope identical in P. marneffei and Aspergillus sp. This immunohistochemical approach could be useful In the diagnosis of a rare disease, penicilliosis marneffei, which proves to be difficult to identify by conventional microscopy.
The spleen of a guinea pig infected with P. marneffei was fixed in formalin and embedded in paraffin. Histologic sections were stained with hematoxylin-eosin, PAS, and GomoriGrocott stain. Other sections were used for immunohistochemistry with the monoclonal antibody EB-AI (Eco-Path Aspergillus, Eco-bio, Genk, Belgium). The method used has been extensively described previously.'^ In short, endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide. Slides were successively incubated with swine serum, EB-AI at a concentration of 20 ng/mL, and with a murine monoclonal anti-rat Ig conjugated to horse radish peroxidase (Mark 3-PO). We used as chromogen the 3amino-9-ethylcarbazole.
Penicillium marneffei is a fungus found in the soil of some countries of Southeast Asia. It has been isolated from bamboo rat burrows. In Vietnam, China, and Thailand, three species of bamboo rats bave been found to be infected with P. marneffei.^ Penicilliosis marneffei mostly affects people who have resided in or have visited Southeast Asian countries. Eess than 40 cases have been reported to date.'"" The disease is quite devastating; it produces cutaneous nodules, pulmonary lesions, osteolytic changes, and splenomegaly. In some patients, immunosuppression appears to be a factor in the acquisition and development of this disease.'''""'' Histologically, P. marneffei is not easy to recognize because conventional bistocbemical stainings such as PAS do not always reveal the fungi. In this study, we tried to identify P. marneffei in paraffin-embedded, formalin-fixed tissue by using a monoclonal antibody directed to Aspergillus galactomannan.''' Aspergillus sp and P. marneffei share some immunodominant epitopes in common,'^ and both respond to oral or parenteral treatment with itraconazole."*
RESULTS
The spleen contained granulomas with discrete necrotic foci. Despite a careful search, PAS stain did not reveal obvious structures of fungi. Conversely, Gomori-Grocott stain and immunolabeling with EB-AI decorated unicellular ovoid yeast-like structures (Figs. 1 and 2). Some of these fungal elements had one cross-wall partitioning them without budding processes.
/
« • •
From the Department of Dermatopathology, CHU du Sart Tilman, Liege, the Division of Diagnostics pasteur, Genk, and the Janssen Research Foundation, Beerse, Belgium. Address for correspondence: J. Arrese Estrada, M.D., Department of Dermatophathology, CHU du Sart Tilman, B-4000, Liege, Belgium.
Figure 1. Penicillium marneffei revealed by Gomori-Grocott stain.
410
}.r
.
identifieation of Peiiicilliiini ntayneffei Ari'ese Esttada et al.
DISCUSSION
The monoclonal antibody EB-AI has been raised against Aspergillus galactomannan."' We had previously shown that this antibody reveals in tissue sections the hyphae form of Aspergillus sp without decorating a large series of other fungi.^''^^ We concluded that EB-A2 was quite specific for Aspergillus sp. Following a recent report showing that Aspergillus sp and P. marneffei had some identical immunodominant epitopes in their external walls,'^ we used in this work EB-Ai in experimental penicilliosis. Our present data reveal that P. marneffei may be identified in tissue sections with this antibody. It is usually easy to distinguish P. marneffei from Aspergillus sp on morphologic grounds. Aspergillus sp appears in tissue sections as mycelial mats composed of radiating hyphae with regular septation and dichrotomic branchings at about 40 degrees. Conversely, the tissue form of P. marneffei develops as a unique yeast-like structure, of 2 to 3 |im in diameter, which is difficult to identify morphologically. They multiply by schizogony and do not form elongated filaments in vivo in deep organs. There are, however, two exceptions to this rule. Long hyphae of P. marneffei may develop in the stratum corneum. The presence of filaments of P. iriarneffei has been reported as an autopsy finding in the lung and liver of an AIDS patient.^- This could reflect a postmortem filamentation due to the low temperature of the body or a unique feature related to AIDS.
Figure 2.
4.
5.
The development of the monoclonal antibody EB-AI, therefore, proves to be a new tool in tbe identification of a rare tropical disease that may emerge as a manifestation of AIDS. The clinical and histologic presentations have been, in some patients, misinterpreted as tuberculosis or histoplasmosis. Tsang et al.'° suggested that penicilliosis marneffei is probably more common than can be judged from the literature. The conventional histologic assessment appears best with methanamine silver impregnation (Gomori-Grocott stain). The use of immtinohistochemistry on histologic sections or on bronchoalveolar lavage fluid could provide the definitive diagnosis.
Drouhet E, Ravisse P, Ave PM, et al. Mycological, ultrastructural and experimental aspects of Penicillium marneffei isolated from a disseminated penicilliosis in AIDS. Bull Soc Fr Mycol Med 1988; 17:77-82.
7.
Deng Z, Rihas JL, Gibson DW, Connor DH. Infections caused hy Penicillium marneffei in China and Southeast Asia: review of eighteen published cases and report of four more Chinese cases. Rev Inf Dis 1988; 10:640-652. Chan JKC, Tsang DNC, Won g KK. Penicillium marneffei in hronchoalveolar lavage fluid. Acta Cytol 1989; 33:523-526. Jayanetra P, Nitiyanant P, Ajello I, et al. Penicilliosis marneffei in Thailand: report of five human cases. Am J Trop Med Hyg 1984; 33:637-644. Tsang DNC, Chan JKC, Lau YT, et al. Penicillium marneffei infection: an underdiagnosed disease? Histopathology 1988; 13:311-318. Yuen WC, Chan YF, Loke SL, et al. Chronic lymphadenopathy cavised by Penicillium marneffei: a condition mimicking tuberculous lymphadenopathy. Br J Surg 1986; 73:1007-1008. Ancelle T, Dupouy-Camet J, Pujol F, et al. Un cas de penicilliose disseminee a Penicilliwn marneffei chez un malade atteint d'un syndrome immunodeficitaire acquis. Presse Med 1988; 17:1095-1096. Peto TEA, Bull R, Mackenzie DWR, et al. Systemic mycosis due to Penicillium marneffei in a patient with antibody to human immunodeficiency virus. J Infect 1988; 16:285-290. Piehl MR, Kaplan RL, Haber MH. Disseminated penicilliosis in a patient with acquired immunodeficiency syndrome. Arch Pathol Lab Med 1988; 112:1262-1264. Stern M, Romana CA, Chovin S, et al. Pencilliose pulmonaire a Penicillium marneffei chez un malade atteint d'un syndrome immunodeficitaire acquis. Presse Med 1989; 18:2067.
8.
9.
10.
11.
REFERENCES
Deng Z, Yun M, Ajello L. Human penicilliosis marneffei and its relation to the bamboo rat (Rhizomys prumosus). J Med Vet Mycol 1986; 24:383-389.
2.
DiSalvo AF, Fickling AM, Ajello L. Infection caused by Penicillium marneffei: description of first natural infection in man. Am J Clin Pathol 1973; 59:259-263. Deng A, Connor D. Progressive disseminated penicilliosis caused by Penieillium marneffei, report of eight cases and differentiation of the causative organism from Histoplastna capsulatum. Am J Clin Pathol 1985; 84:323-327.
3.
13.
14.
15.
411
So SY, Chau PY, Jones BM, et al. A case of invasive penicilliosis in Hong Kong with immunologic evaluation. Am Rev Resp Dis 1985; 131:662-665. McGinnis MR. Progressive disseminated penicilliosis caused hy Penicillium marneffei - unfortunate omission, (letter to the editor). Am J Clin Pathol 1986; 85:529.
6.
12.
1.
Penicillium marneffei revealed by the monoclonal
antibody EB-AI.
International Journal of Dermatology Vol. 31, No. 6, June 1992
16. Goris A, Sarfati J, Diaquin M, et al. Monoclonal antihodies against Aspergillus galactomannan: characterization and application in immunoelectron microscopy. Abstract 4th European Congres of Clinical Microbiology, Nice 1989. p.8O. 17. Notermans S, Veeneman GH, Van Zuylen CWFM, et al. (1-5) linked heta-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicillium and Aspergillus species. Mol Immunol 1988; 25:975-979. 18. Van Cutsem J. Fungal models in immunocompromised animals. In: Van Den Bossche H, Mackenzie DWR, Cauwenbergh G, et al., eds. Mycoses in AIDS patients. New York: Plenum Press, 1990:207.
19. Arrese Estrada J, Stynen D, Goris A, Pierard GE. Identification immunohistochimique des Aspergillus par l'anticorps monoclonal EB-AI. Ann Pathol 1990; 10:198-201. 20. Arrese Estrada J, Stynen D, Goris A, et al. Immunohistochemical identification of Aspergillus sp. and P. marneffei infections. J Invest Dermatol 1991; 96:639. 21. Arrese Estrada J, Rurangirwa A, Pierard GE. Estudio morfometrico del Aspergillus en lesiones humanas. Rev Ibero Am Micologia (in press). 22. Hulshof CMJ, Van Zanten RAA, Sluiters LH, et al. Penicillium marneffei infection in an AIDS patient. Eur J Clin Microbiol Infection Dis 1990; 9:370.
Decubitus Ulcers Paget did not specifically mention pressure sores in his list of postoperative complications, probably because patients undergoing surgery in tbe nineteenth century either did not survive long enough to get them, or were too young and otherwise healthy to be at risk. However, like Hunter and Charcot, he regarded management of pressure sores as essential to the practice of surgery. He included bedsores in the series of lectures given at St. Bartholomew's Hospital, London, and the Royal College of Surgeons that formed the basis of modern medical student teaching and led to the requirement of a medical degree for the practice of surgery in 1866. Having spent the first 7 years of his London career studying pathology, Paget began his lecture with a pathological definition of a bedsore as "the sloughing and mortification or death of a part produced by pressure". He identified certain "forerunners": "inflammation affecting prominent parts", usually the sacrum, the posterior superior spines of the ilium, the trochanters, and the ends of the spines of the vertebrae. That he did not mention the ischial tuberosities is probably because ill patients in his day did not sit in chairs. He described other signs of pressure damage as "pale or white patches" or "purple or yellow discoloration from the extravasation of blood or bloody fluid". "Sloughing follows these in the skin and subcutaneous tissue and fat. These latter die before the skin as sloughing proceeds faster in them, so when the skin comes away, the place formerly occupied by these tissues is empty." These careful ohservations anticipate later studies in animals and tissue viability measurements in man, which have shown that interstitial sores occur primarily in the deep tissues surrounding an underlying bony prominence. Pressure differences which cause capillary distortion and cut off the blood supply are greater in the region of a hard object such as a bone than in the skin. Consequently, deep pressure sores characteristically have overhanging edges, and apparently small skin lesions may interconnect by subcutaneous sinuses extending for many centimetres under intact skin. Paget observed, these sinuses result from breakdown of dead tissue produced during the original ischaemic episode; they are not, as is so commonly asserted, due to pressure or infection from a superficial sore spreading into the deeper layers. Prom Bliss MR. Acute pressure area care: Sir James Paget's legacy. Lancet 1992; 339:221-223. 412
REPORT
IMMUNOHISTOCHEMICAL IDENTIFICATION OE PENICILLIUM MARNEFFEI BY MONOCLONAL ANTIBODY JORGE ARRESE ESTRADA, M.D., DIRK STYNEN, PH.D., JAN VAN CUTSEM, PH.D.,
CLAUDINE PIERARD-ERANCHIMONT, M.D., AND GERALD E. PIERARD, M.D.
Abstract
Materials and Methods
We present an experimental study on the immunohistochemical identification of Penicillium marneffei in paraffinembedded, formalin-fixed tissue. The monoclonal antibody EB-A1 detects a specific galactomannan that appears to have at least one epitope identical in P. marneffei and Aspergillus sp. This immunohistochemical approach could be useful In the diagnosis of a rare disease, penicilliosis marneffei, which proves to be difficult to identify by conventional microscopy.
The spleen of a guinea pig infected with P. marneffei was fixed in formalin and embedded in paraffin. Histologic sections were stained with hematoxylin-eosin, PAS, and GomoriGrocott stain. Other sections were used for immunohistochemistry with the monoclonal antibody EB-AI (Eco-Path Aspergillus, Eco-bio, Genk, Belgium). The method used has been extensively described previously.'^ In short, endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide. Slides were successively incubated with swine serum, EB-AI at a concentration of 20 ng/mL, and with a murine monoclonal anti-rat Ig conjugated to horse radish peroxidase (Mark 3-PO). We used as chromogen the 3amino-9-ethylcarbazole.
Penicillium marneffei is a fungus found in the soil of some countries of Southeast Asia. It has been isolated from bamboo rat burrows. In Vietnam, China, and Thailand, three species of bamboo rats bave been found to be infected with P. marneffei.^ Penicilliosis marneffei mostly affects people who have resided in or have visited Southeast Asian countries. Eess than 40 cases have been reported to date.'"" The disease is quite devastating; it produces cutaneous nodules, pulmonary lesions, osteolytic changes, and splenomegaly. In some patients, immunosuppression appears to be a factor in the acquisition and development of this disease.'''""'' Histologically, P. marneffei is not easy to recognize because conventional bistocbemical stainings such as PAS do not always reveal the fungi. In this study, we tried to identify P. marneffei in paraffin-embedded, formalin-fixed tissue by using a monoclonal antibody directed to Aspergillus galactomannan.''' Aspergillus sp and P. marneffei share some immunodominant epitopes in common,'^ and both respond to oral or parenteral treatment with itraconazole."*
RESULTS
The spleen contained granulomas with discrete necrotic foci. Despite a careful search, PAS stain did not reveal obvious structures of fungi. Conversely, Gomori-Grocott stain and immunolabeling with EB-AI decorated unicellular ovoid yeast-like structures (Figs. 1 and 2). Some of these fungal elements had one cross-wall partitioning them without budding processes.
/
« • •
From the Department of Dermatopathology, CHU du Sart Tilman, Liege, the Division of Diagnostics pasteur, Genk, and the Janssen Research Foundation, Beerse, Belgium. Address for correspondence: J. Arrese Estrada, M.D., Department of Dermatophathology, CHU du Sart Tilman, B-4000, Liege, Belgium.
Figure 1. Penicillium marneffei revealed by Gomori-Grocott stain.
410
}.r
.
identifieation of Peiiicilliiini ntayneffei Ari'ese Esttada et al.
DISCUSSION
The monoclonal antibody EB-AI has been raised against Aspergillus galactomannan."' We had previously shown that this antibody reveals in tissue sections the hyphae form of Aspergillus sp without decorating a large series of other fungi.^''^^ We concluded that EB-A2 was quite specific for Aspergillus sp. Following a recent report showing that Aspergillus sp and P. marneffei had some identical immunodominant epitopes in their external walls,'^ we used in this work EB-Ai in experimental penicilliosis. Our present data reveal that P. marneffei may be identified in tissue sections with this antibody. It is usually easy to distinguish P. marneffei from Aspergillus sp on morphologic grounds. Aspergillus sp appears in tissue sections as mycelial mats composed of radiating hyphae with regular septation and dichrotomic branchings at about 40 degrees. Conversely, the tissue form of P. marneffei develops as a unique yeast-like structure, of 2 to 3 |im in diameter, which is difficult to identify morphologically. They multiply by schizogony and do not form elongated filaments in vivo in deep organs. There are, however, two exceptions to this rule. Long hyphae of P. marneffei may develop in the stratum corneum. The presence of filaments of P. iriarneffei has been reported as an autopsy finding in the lung and liver of an AIDS patient.^- This could reflect a postmortem filamentation due to the low temperature of the body or a unique feature related to AIDS.
Figure 2.
4.
5.
The development of the monoclonal antibody EB-AI, therefore, proves to be a new tool in tbe identification of a rare tropical disease that may emerge as a manifestation of AIDS. The clinical and histologic presentations have been, in some patients, misinterpreted as tuberculosis or histoplasmosis. Tsang et al.'° suggested that penicilliosis marneffei is probably more common than can be judged from the literature. The conventional histologic assessment appears best with methanamine silver impregnation (Gomori-Grocott stain). The use of immtinohistochemistry on histologic sections or on bronchoalveolar lavage fluid could provide the definitive diagnosis.
Drouhet E, Ravisse P, Ave PM, et al. Mycological, ultrastructural and experimental aspects of Penicillium marneffei isolated from a disseminated penicilliosis in AIDS. Bull Soc Fr Mycol Med 1988; 17:77-82.
7.
Deng Z, Rihas JL, Gibson DW, Connor DH. Infections caused hy Penicillium marneffei in China and Southeast Asia: review of eighteen published cases and report of four more Chinese cases. Rev Inf Dis 1988; 10:640-652. Chan JKC, Tsang DNC, Won g KK. Penicillium marneffei in hronchoalveolar lavage fluid. Acta Cytol 1989; 33:523-526. Jayanetra P, Nitiyanant P, Ajello I, et al. Penicilliosis marneffei in Thailand: report of five human cases. Am J Trop Med Hyg 1984; 33:637-644. Tsang DNC, Chan JKC, Lau YT, et al. Penicillium marneffei infection: an underdiagnosed disease? Histopathology 1988; 13:311-318. Yuen WC, Chan YF, Loke SL, et al. Chronic lymphadenopathy cavised by Penicillium marneffei: a condition mimicking tuberculous lymphadenopathy. Br J Surg 1986; 73:1007-1008. Ancelle T, Dupouy-Camet J, Pujol F, et al. Un cas de penicilliose disseminee a Penicilliwn marneffei chez un malade atteint d'un syndrome immunodeficitaire acquis. Presse Med 1988; 17:1095-1096. Peto TEA, Bull R, Mackenzie DWR, et al. Systemic mycosis due to Penicillium marneffei in a patient with antibody to human immunodeficiency virus. J Infect 1988; 16:285-290. Piehl MR, Kaplan RL, Haber MH. Disseminated penicilliosis in a patient with acquired immunodeficiency syndrome. Arch Pathol Lab Med 1988; 112:1262-1264. Stern M, Romana CA, Chovin S, et al. Pencilliose pulmonaire a Penicillium marneffei chez un malade atteint d'un syndrome immunodeficitaire acquis. Presse Med 1989; 18:2067.
8.
9.
10.
11.
REFERENCES
Deng Z, Yun M, Ajello L. Human penicilliosis marneffei and its relation to the bamboo rat (Rhizomys prumosus). J Med Vet Mycol 1986; 24:383-389.
2.
DiSalvo AF, Fickling AM, Ajello L. Infection caused by Penicillium marneffei: description of first natural infection in man. Am J Clin Pathol 1973; 59:259-263. Deng A, Connor D. Progressive disseminated penicilliosis caused by Penieillium marneffei, report of eight cases and differentiation of the causative organism from Histoplastna capsulatum. Am J Clin Pathol 1985; 84:323-327.
3.
13.
14.
15.
411
So SY, Chau PY, Jones BM, et al. A case of invasive penicilliosis in Hong Kong with immunologic evaluation. Am Rev Resp Dis 1985; 131:662-665. McGinnis MR. Progressive disseminated penicilliosis caused hy Penicillium marneffei - unfortunate omission, (letter to the editor). Am J Clin Pathol 1986; 85:529.
6.
12.
1.
Penicillium marneffei revealed by the monoclonal
antibody EB-AI.
International Journal of Dermatology Vol. 31, No. 6, June 1992
16. Goris A, Sarfati J, Diaquin M, et al. Monoclonal antihodies against Aspergillus galactomannan: characterization and application in immunoelectron microscopy. Abstract 4th European Congres of Clinical Microbiology, Nice 1989. p.8O. 17. Notermans S, Veeneman GH, Van Zuylen CWFM, et al. (1-5) linked heta-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicillium and Aspergillus species. Mol Immunol 1988; 25:975-979. 18. Van Cutsem J. Fungal models in immunocompromised animals. In: Van Den Bossche H, Mackenzie DWR, Cauwenbergh G, et al., eds. Mycoses in AIDS patients. New York: Plenum Press, 1990:207.
19. Arrese Estrada J, Stynen D, Goris A, Pierard GE. Identification immunohistochimique des Aspergillus par l'anticorps monoclonal EB-AI. Ann Pathol 1990; 10:198-201. 20. Arrese Estrada J, Stynen D, Goris A, et al. Immunohistochemical identification of Aspergillus sp. and P. marneffei infections. J Invest Dermatol 1991; 96:639. 21. Arrese Estrada J, Rurangirwa A, Pierard GE. Estudio morfometrico del Aspergillus en lesiones humanas. Rev Ibero Am Micologia (in press). 22. Hulshof CMJ, Van Zanten RAA, Sluiters LH, et al. Penicillium marneffei infection in an AIDS patient. Eur J Clin Microbiol Infection Dis 1990; 9:370.
Decubitus Ulcers Paget did not specifically mention pressure sores in his list of postoperative complications, probably because patients undergoing surgery in tbe nineteenth century either did not survive long enough to get them, or were too young and otherwise healthy to be at risk. However, like Hunter and Charcot, he regarded management of pressure sores as essential to the practice of surgery. He included bedsores in the series of lectures given at St. Bartholomew's Hospital, London, and the Royal College of Surgeons that formed the basis of modern medical student teaching and led to the requirement of a medical degree for the practice of surgery in 1866. Having spent the first 7 years of his London career studying pathology, Paget began his lecture with a pathological definition of a bedsore as "the sloughing and mortification or death of a part produced by pressure". He identified certain "forerunners": "inflammation affecting prominent parts", usually the sacrum, the posterior superior spines of the ilium, the trochanters, and the ends of the spines of the vertebrae. That he did not mention the ischial tuberosities is probably because ill patients in his day did not sit in chairs. He described other signs of pressure damage as "pale or white patches" or "purple or yellow discoloration from the extravasation of blood or bloody fluid". "Sloughing follows these in the skin and subcutaneous tissue and fat. These latter die before the skin as sloughing proceeds faster in them, so when the skin comes away, the place formerly occupied by these tissues is empty." These careful ohservations anticipate later studies in animals and tissue viability measurements in man, which have shown that interstitial sores occur primarily in the deep tissues surrounding an underlying bony prominence. Pressure differences which cause capillary distortion and cut off the blood supply are greater in the region of a hard object such as a bone than in the skin. Consequently, deep pressure sores characteristically have overhanging edges, and apparently small skin lesions may interconnect by subcutaneous sinuses extending for many centimetres under intact skin. Paget observed, these sinuses result from breakdown of dead tissue produced during the original ischaemic episode; they are not, as is so commonly asserted, due to pressure or infection from a superficial sore spreading into the deeper layers. Prom Bliss MR. Acute pressure area care: Sir James Paget's legacy. Lancet 1992; 339:221-223. 412
