PATHOLOGY RESEARCH AND PRACTICE e Urban & Fischer Verlag http://www.urbanfischer.deljournalslprp

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Detection of the 20-kDa Virulence-associated Antigen of Rhodococcus equi in Malakoplakia-like

Lesion in Pleural Tissue Obtained from an AIDS Patient*

Adele Caterino-de-Araujo', Elizabeth de los Santos-Fortuna ', Monica Cristina Zandona-Meleiro', Edenilson Eduardo Calore2

and Nilda Maria Perez Calore2

'Immunology Department, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil ; 'Pathology Section, Emilio Ribas Institute, Sao Paulo, Sp, Brazil

Summary

A malakoplakia-like lesion was detected in a pleural biopsy from an AIDS patient presenting clinical and ra­diologic features of pneumonia. Cultures of bron­choalveolar lavage and pleural fluid evidenced Rhodococcus equi as the causative agent of pleuro-pul­monary infection. Immunochemical characterization of the R. equi isolate showed the presence of a strain simi­lar to the ATCC 33704 reference strain presenting the capsular antigen of serotype 4, and the intermediate vir­ulence-associated antigen of 20-kOa. Histopathology of the patient's pleural biopsy showed plaques of macrophages interspersed with lymphocytes, and intra­cytoplasmic cocci and bacilli in macrophages, which were variably acid-fast positive. Immunohistochemistry of cocci, bacilli and their degradation products resulted strongly positive when stained with a mouse monoclon­al antibody (MAb) produced against the 20-kDa anti­gen. This finding could have important implications for the pathogenicity of R. eqlli for human beings. since we do not know yet all the factors involved in the forma­tion of malakoplakia. Indeed , the results obtained in the present study, taken together with the results obtai ned for pigs inoculated with R. equi strains of intermediate virulence (Madarame et al. 1998), raise the possibility that most strains presenting the 20-lOa antigen may be

*This work was partial ly supported by Funda~ao de Amparo ~ Pesquisa do Estado de Sao Paulo (FAPESP), grant number 97/02866-0.

Pathol. Res. Pract. 196: 32 1-327 (2000)

capable of inducing malakoplakia. If this hyphothesis is confirmed by immunohistochemical analysis of human pu lmonary malakoplakia cases due to R. equi, the de­tection of this antigen may be extremely helpful in the diagnosis and treatment of such patients. This is the first report of R. eqlli infection in human beings that sug­gests a relationship between pleural malakoplakia and the viru lence-associated antigen of 20-kOa.

Key words: RhodocoCCllS equi - Malakoplakia - AIDS - Virulence-associated antigen of 20-kOa - Immuno­histochemistry - Macrophages

Introduction

Malakoplakia is a chronic granulomatous inflamma­tion characterized by accumulat ion of granular histio­cytes, possibly containing intracytoplasmic basophilic inclusions (Michaelis Gutmann bodies), at various body sites, mainly in the lower urinary tract {S/. It was first described by Michaelis and Gutmann in 1902 [l3} and named "malakoplakia" by von Hansemann in 1903 {31}. Until 1996, approximate ly 400 cases had been re-

Address for correspondence: Adele Caterino-de-Araujo, In­stituto Adolfo Lutz, Av. Dr. Arnatdo 355. t I andar, 01246-902. Sao Paulo, SP, Brazil. Phone: ++55- 11 -30~ I0111 ext. 2065, Fax: ++55-11-8533505. E-mait: (catcrinu@usp.br) (caterino@ ial.sp.gov.br)

0344-0338/2000/196/5-32 1 $12.00/0

322 . A. Caterino-de-Araujo et al.

ported, most of them associated with Escherichia coli infection [30J. Malakoplakia in those cases was associ­ated with immunosuppression of patients with organ transplantation, hematopoietic malignancy, or alco­holism. Pulmonary malakoplakia is rare, and was most­ly detected in Human Immunodeficiency Virus (HIV)­infected patients, in which Rhodococcus equi could be isolated from respiratory specimens [11j.

R. equi is a Gram-positive coccobacillus present in soil which causes bronchopneumonia, enteritis or mesenteric adenitis in horses, cattle, swine and sheep [16]. In humans it is responsible for necrotizing pneu­monia with or without cavitation or spindle cell prolif­eration [l6}. R. equi with at least two levels of viru­lence were detected among AIDS-infected patients: R. equi expressing the virulence-associated antigen of 15-to 17-kDa (similar to isolates from foals) , and R. equi expressing the intermediate virulence-associated anti­gen of 20 kDa (similar to isolates from pigs). However, some cases of rhodococcal infection in AIDS were due to strains considered avirulent [3, 18, 22}.

In veterinary medicine, malakoplakia is not found in lung tissues from infected foals, even when these ani­mals are infected with virulent R. equi strains (present­ing the 15- to 17-kDa virulence-associated antigen). On the other hand, two cases of spontaneous malakoplakia have been reported in pigs, one of which in lymph nodes from a slaughtered pig [8}, and the other one sys­temically disseminated in a breeding pig [27}. Cuta­neous malakoplakia was also induced experimentally in pigs by subcutaneous inoculation of intermediate viru­lent R. equi strains, in which immunohistochemical analysis of the lesions showed the presence of the 20-kDa antigen in Michaelis Gutmann (MG) bodies, pro­viding evidence of the involvement of R. equi and its virulence antigens in the process of malakoplakia [12}.

Some of us previouly described one case of pleural malakoplakia-like lesion in an AIDS patient in which R. equi could be isolated from pleural tluid and bron­choalveolar lavage, and suggested the use of histo­chemistry for a rapid and accurate diagnosis of rhodococcal infection in the lung [2}. In the present study we have used immunohistochemical analysis of the same biopsy material to search for an R. equi strain related to malakoplakia formation in humans. We also characterized immunochemically the R. equi isolated from this patient, and compared it with R. equi refer­ence strains.

Materials and Methods

Case report

The patient was a 23-year-old heterosexual man, an intra­venous drug user for the last three years, who came to Institu-

to de Infectologia Emilio Ribas (HER), Sao Paulo, complain­ing of bilateral chest pain and cough for 60 days, having had a weight loss of 10.0 kg in the last 16 days. He was treated with penicillin and second generation cephalosporin without suc­cess. A chest roentgenogram showed an abnormal opacity of the right lower lobe. The WBC was 15,500 (77% of neu­trophils). His haemoglobin level was 11.0 gil and the platelet blood count was normal. Biochemical screening showed only a slight decrease in albumin level (1.9 mgldl). Serology for HIV was performed and resulted positive. At this time he pre­sented also oral candidiasis. He was submitted to pleural drainage of the right hemithorax and to a pleural biopsy. The pleural effusion was characteristic of empyema. Rhodococcl4S equi grew in culture. The patient was treated with ery­thromycin for 14 days with success.

Bacterial strains

The clinical bacterial isolate was cultured from bron­choalveolar lavage and pleural fluid of the patient and sent to Instituto Adolfo Lutz (IAL) for analysis. The presence of R. equi was confirmed at the Culture Collection Sector on the basis of Gram-positive coccobacilli, salmon-pink mucoid colonies, oxidase negativity, CAMP test positivity, and also on the basis of the results of the API Corynebacterium test system (Biomerieux, Marcy l'Etoile, France). The isolate was lyophilized and stored with the identification number IAL-2043.

R. equi ATCC 33701 and ATCC 33704 were used as refer­cnce strains because their plasmid content and virulence level have already been described (26, 28/.

All strains were used in antigen preparations for serotyping and immunoblotting analysis.

Antibodies

Monoclonal antibodies (MAbs) against the 15- to 17-kDa (MAb, I OG5) /2/ and 20-kOa antigens [25J were provided by Or. S. Takai and used to identify virulence-associated antigens in immunoblotting and immunohistochemistry.

Seven rabbit sera (1-7) which react with the capsular anti­gens of R. equi were provided by Dr. J. Prescott and were used for serotyping the R. equi isolate [15].

Serum from a foal naturally infected with R. equi, and sera from the patient were analysed by immunoblot, along with serum from another infected human patient.

Strain characterization

R. equi was grown at 37 "C on Miiller Hinton agar (Oifco) for 48h and subsequently harvestcd in physiologic solution (0.9% NaCl). This suspension was maintained at 37 "C, under shaking, overnight, and then spun at 4,000 rpm. The capsular antigens in the supernatants were submitted to immunodiffu­sion in agar gel, as previously described {lSI.

To compare the major antigenic components of the human R. equi isolate with those of R. equi reference strains, whole­cell antigens prepared by harvesting bacteria grown at 38 °C for 48 h from brain heart infusion broth (Oifco), and solubi­lized in sodium dodecyl sulphate (50S) reducing buffer, were submitted to SOS-polyacrylamide gel electrophoresis. The

20-kDa Antigen of Rhodococcus equi and Malakoplakia-like Lesion in AIDS . 323

components were subsequently elcctrotransferred to nitrocel­lulose sbeets for immunoblolling. Tbe immunoblOl analysis of the isolate was conducted according to the technique previous­ly established in the Immunology Department of IAL {I 8, 33].

Histopathological and immunohistochemical procedures

The histopathology of the pleural tissue was conducted in paraffin wax-embedded sections submitted to hematoxyl­eosin staining and to Fite's method for acid-fast organisms as previously described (2/. Immunohistochemical reactions were performed according to Hsu et al. 1981 (9), using as pri­mary antibodies, mouse monoclonal antibodies against the 15- to 17-kDa antigen and 20-kDa antigen. diluted I: I 00 and I: I 0 ,000 in phosphate buffered saline, pH 7.4, with 1% bovine serum albumin (PBS-BSA). Briefly. slices of paraffin embedded sections were treated with I % aqueous hydrogen peroxide and methanol (Sigma) for 20 minutes, and then incu­bated with the primary antibodies for 24 hours at 4 °C in a moist chamber. The slices were then incubated sequentially with biotinylated secondary antibody (Sigma) for 30 minutes at 37 °C, with the streptavidin-biotin-peroxidase complex (Sigma) for 30 minutes at 37 °C, and finally with aqueous di­aminobenzidine solution (40 mg/ml) and 0.1 % (v/v) hydrogen peroxide (Sigma) in PBS. The slices were counterstained with hematoxylin, dehydrated, cleared and mounted with entellan. The incubation step with primary antibodies was skipped for the negative controls. In addition. to assure the specificity of the primary antibodies in the immunohistochemical reaction for R. equi , the same procedure was performed using paraffin wax-embedded sections of samples diagnosed as tuberculosis and other non-specific bacterial diseases.

Results

The results obtained in the characterization of the R. equ; isolate showed a strain similar to the ATCC 33704 reference strain, presenting the capsular antigen of type 4 and the intermediate virulence-associated anligen of

33101 33704

1 2 3 . , , ' 3 . , , , 3

20-kDa. Immunoblotting showed reactivity of the in­fected foal serum with several antigens present in this isolate, although reactivity to low molecular weight antigens could not be detected (Fig. J, strain IAL-2043, lane I). On the other hand, serum samples from infected human patients strongly reacted with the low molecular weight antigens (Fig. 1, strain IAL-2043, lanes 4-5), a reactivity similar to that of the MAb to the 20-kDa anti­gen (Fig. 1, strain IAL-2043, lane 3).

Immunoblot analysis of reference strains confirmed the presence of the 15- to 17-kDa anligen in the ATCC 33701 strain (Fig. 1, strain ATCC 33701, lane 2), and of the 20-kDa antigen in the ATCC 33704 strain (Fig. I, strain ATCC 33704, lane 3). The foal serum sample tested with these reference strains demonstrated a better reactivity of antibodies to the antigens present in the ATCC 33701 strain (Fig. I, strains 33701 and 33704, lanes I). In contrast, when infected human serum sam­ples were analysed by immunoblol, a better seroreactiv­ity to several antigens, including the 20-kDa antigen, was observed in the ATCC 33704 reference strain (Fig. I, strains ATCC 3370 I and 33704, lanes 4-5).

The results of the histopathological analysis of the pleural biopsy from the patient revealed a malako­plakia-like lesion in the pleural ti ssue (Fig. 2A), with a large number of gram-positive and variable acid-fast positive coccobacilli in macrophages (Fig. 2B and C) /2J. Reactivity to the virulence-associated antigen of 20-kDa could be detected by immunohistochemistry: this reactivity was widely spread among macrophages , in which coccobacilli and their fragments resulted posi­tive (Fig. 3A and B). Stains around the whole bacteria membrane were also observed (Fig. 3C). No reactivity was detected by immunohistochemistry when the MAb to the 15- to 17 -kDa antigen was used, or when tissue samples from patients presenting tuberculosis and other bacterial diseases were tested.

. ,

Fig. I. Immunoblot profiles of whole cell R. equi antigens from ATCC 33701 , ATCC 33704. and IAL-2043 strains, when tested with foal serum (lane I), MAbs to the 15- to 17-kDa and 20-kDa (lanes 2 and 3), and human sera (lanes 4 and 5). Molecular size markers of 97.4, 66.2,45.0, 31.0, 21.5, and 14.4 kDa are indicated on the right. The arrow on the left points at the bands for the 15- to 20-kDa pro­teins.

324 ' A, Caterino-de-Araujo et aJ.

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Fig. 2. Pleural biopsy sections. A: Dense colleclion of macrophages interspersed with some lymphocytes, with the appearenee of malakoplakia (Fite's stain, xIOO), B and C: Higher magnification of pleural malakoplakia presenting fine­ly granular and vacuolated macrophages, and variably acid­fast positive coccobacilli (Fite's stain, x400 and xl ,000).

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20-kDa Amigen of Rhodococcus equi and Malakoplaki a- like Lesion in AIDS 325

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Fig. 3. Immunohistochemistry of pleural malakoplakia, A: Numerous R. equi and their degradation products stained with the monoclonal antibody to the 20·kDa antigen of the R. equi strain of intermediate virulence (small star). The adjacent con­nective tissue stained negatively (big star) (Labeled avidin-bi­otin method, hematoxylin eosin counterstain, x40). B: Sheets of histiocytes, many containing R. equi stained positively by immunohistochemistry (Labeled avidin· biotin method using MAb to the 20-kOa antigen of R. equi, hematoxylin eosin counterstain, x400). C: Higher magnification showing posi­tivity on the cellular membrane of bacteria (arrows) (Labeled avidin·biotin method using MAb to the 20-kDa antigen of R, equi, hematoxylin eosin counterstain, x 1,000),

326 . A. Caterino-de-Araujo et aJ.

Discussion

Pulmonary infection by R. equi in HIV-infected pa­tients has been described [6, 20, 29/, and in several cases this infection was associated with malakoplakia [7, 11,14, 19- 21]. Usually, the diagnosis of R. equi in­fection is performed by bacterial isolation and subse­quent identification in blood or bronchoalveolar lavage, but these procedures take several days. Alterna­tive methods for the detection of R. equi have been taken into consideration, and the polymerase chain re­action (PCR) stands out as a fast, sensitive and accuratc technique to identify the 15- to 17-kDa virulence-asso­ciated antigens {24 J and the R. equi species {l]. How­ever, these methodologies are expensive and need so­phisticated laboratories in order to avoid contamina­tion.

In the last few years the diagnosis of malakoplakia by fine needle aspiration and lung tissue biopsy was considered of some help {II, 17,21, 32J, and specific stains for acid-fast bacteria and for detecting Michaelis Gutmann (MG) bodies have also been used {2, 10, 11 , 32]. The number of MG bodies varies from case to case and appears to be inversely proportional to the number of bacteria in the tissue lesion {4, 10]. We found in the present study large numbers of bacteria and no MG bodies in tissue (Fig. 2).

Recently, an association between the 20-kDa viru­lence-associated antigen of R. equi and malakoplakia was demonstrated experimentally in pigs inoculated with R. equi of intermediate virulence, using immuno­histochemical analysis {l2]. On the basis of this re­port, we may speculate about a possible role for the 20-kDa antigen in the malakoplakia process in human beings. Since we had stored the bronchoalveolar lavage isolate and the pleural biopsy specimen from an AIDS patient in whom a malakoplakia-like lesion had been detected, we compared the antigenic pattern of this patient's isolate with two R. equi reference strains, and searched for the virulence-associated antigens in the lesion.

The results obtained by gel agar immunodiffusion and immunoblotting confirmed the similarity between the human isolate and the ATCC 33704 reference strain (Fig. I), indicating that pigs and their environment are possible sources of R. equi infection for humans. Im­munohistochemistry confirmed the association between the 20-kDa antigen and malakoplakia.

This is the first time that an association between the 20-kDa virulence-associated antigen and malakoplakia has been observed in humans. This finding, taken to­gether with the same finding in pigs, raises the possibil­ity that most R. equi strains presenting the 20-kDa anti­gen could induce malakoplakia. We suggest a search for the antigen in malakoplakia cases caused by R. equi de­tected around the world and described in the literature.

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Received: April 12,1999 Accepted in revised version: November 9. 1999