Bronchoscopy Specimens in Adults with AIDS

Bronchoscopy Specimens in Adults with AIDS* Comparative Yields of Cytology, Histology and Culture for Diagnosis of Infectious Agents C. Michael Weldon-Linne, M.D.;t Douglas P. Rhone, M .D.;:t: and Renee Bourassa§

Bronchoscopy specimens from 183 lmown/suspected acquired immunodeficiency syndrome patients were evaluated for pathogens. In each case, transbronchial biopsies were evaluated and bronchoalveolar lavage material was cultured for viruses, fungi and mycobacteria and examined cytologically. A specimen was considered positive for a pathogen if detected by any one of the methods (TBB or BALC or culture). BALC was more sensitive for Pneumocyms carinii than TBB (90 or'92 vs 61 of 80 cases). TBB and BALC had poor sensitivities for cytomegalovirus detection (six of 79 and ten of 91 cases, respectively): 80 of 91 CMV cases were detected by culture only. Nineteen of 26 MB cases were positive by culture only: BALC and TBB

detected on1y three of 26 and five of 23 cases, respectively. Three cryptococcosis cases were detected by culture on1y. One coccidioidomycosis case was positive by BALC and culture. Culture and BALC in combination detected 212 of 216 all significant pathogens. We believe that TBB is not routinely necessary in AIDS-related bronchoscopies in the absence of suspicion of neoplasia. (Chat 1990; 98:!4-28)

the appearance, in 1981, of the first reports Since describing AIDS, it has become evident that

1986 through July 1987, were analyzed retrospectively. The patient population consisted of 126 adults (120 men, six women), all of whom were either known to have AIDS {as currently defined by the Centers for Disease Control"), were in an epidemiologic group at risk fur AIDS, 11 or were seropositive for human immunodeficiency virus. Bronchoscopy was performed for evaluation of pulmonary signs and/or symptoms, including but not limited to dyspnea, cough, arterial hypoxemia, abnormal chest roentgenogram, and abnormal pulmonary gallium scan; 38 of the 126 patients had two or more bronchoscopies performed. Bronchoalveolar lavage was performed during all183 procedures. The standard procedure consisted of instilling 100 to 150 ml of normal saline solution, in small aliquots; aspirated returns from most patients were in the range of 50 to 75 mi. TBBs were performed at 148 of the 183 bronchoscopies. Mycobacterial, fungal, and viral cultures were performed on all but three of the 183 BAL specimens. For MB isolation, BAL fluid was diluted 1:1 with sodium tri-basic phosphate digestant and agitated for 15 minutes. The resultant specimen was neutralized to a pH of 5.5 with 2N hydrochloric acid and concentrated by centrifugation for 15 minutes at 3000 rpm. The sediment was vortexed and inoculated onto Lowenstein-Groft, American Thoracic Society, and 7H10 media slants. Air dried, heat-fixed smears of sediment were stained with Kinyoun's acid-fast procedure and examined. For fungus isolation, BAL fluid was centrifuged at 2,500 rpm for 15 minutes. Sediment was inoculated onto Sabouraud's and Mycosel agar and supernatant fluid was inoculated into brain-heart infusion broth. For virus isolation, BAL fluid was diluted 1:1 with viral transport media, vortexed, and centrifuged at 2"C fur live minutes at 2,000 rpm . Supernatant fluid was inoculated into tubes containing the following cell monolayers: HEp-2 heteroploid cells, A549 heteroploid cells, MRCS fibroblasts, human foreskin fibroblasts, and primary rhesus monkey kidney epithelial cells. Repassage was performed on all tubes demonstrating CPE . Confirmation was performed on CPE-positive repassed specimens using direct fluo-

12 •

pulmonary complications are extremely common in AIDS patients and that these complications are responsible for a significant percentage of both the morbidity and mortality seen in such individuals.3-S Opportunistic infections comprise the overwhelming majority of AIDS-related pulmonary complications. 4 •5 While many recent studies have established the efficacy of bronchoscopy in the diagnosis of pulmonary infections in AIDS,s- 10 such reports have not focused upon the individual sensitivities of the various diagnostic modalities available to the bronchoscopist. The purpose of the present study was to evaluate the relative sensitivities of BALC, TBB, and culture for the detection of infectious pathogens in patients with AIDS-related pulmonary infections. METHODS Results from specimens obtained at 183 consecutive bronchoscopies, perfOrmed at Illinois Masonic Medical Center from January *From the Sections of Clinical Microbiology, and Virology and Cytology, Department of Pathology, Illinois Masonic Medical Center, and the Department of Pathology, University of Illinois College of Medicine at Chicago. tDirector, Section of Clinical Microbiology and Virology, and Assistant Clinical Professor. :!:Chairman and Associate Professor. §Supervisor, Section of Cytology. Manuscript received October 27; revision accepted December 7. Reprint requests: Dr. ~ldon-Unne, lbthology, Illinois Masomc Medlcal Center; Chicago 60637

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TBB = transbroncbial biopsy; MB =mycobacteria; BALC = BAL cytology; PC= Pneumoc,atia ctJrinii; CMV =cytomegalovirus; 1DV =human immuuode6ciencr.. vi-

rus; BAL=broocboalveolar lavage; CPE=cytopathic enect; MTB=M tuberculoria; MAI=M ~; MK=M Armacaaii; NTMB = nontuberculous mycobacteria

Bronchaecopy Specimens In Adulls wllh AIDS {Wtlldan-Unne, Rhone, Bourasaa)

Table 1-ln/ectioua Pathogena IJemon.trated in Bronchoacopic Specimens nom lbtienta with AIDS• Pneumocystis cariniit Cytomegalovirus Mycobacterium avium-intraceUulare Mycobacterium tuberculosis Mycobacterium kansasii Cryptococcus sp Herpes simplex virus Coccidioide.v immitis Acid-fast bacilli-culture negative

92(50.3%) 91 (49.7%) 17 (09.3%) 4(02.3%) 4 (02.3%) 3(01.6%) 3(01.6%) 1 (00.5%) 1 (00.5%)

•Percent of specimens positive in parentheses. tNote: 141 of the 183 bronchoscopies perfonned yielded at least one infectious pathogen; 76 had one only; 55 had two pathogens; ten had three different pathogens.

rescent antibody techniques. Hemadsorption was perfonned on rhesus monkey kidney monolayers at ten to 14 days to detect hemadsorbing viruses. Cytologic examination was perfonned on all 183 BAL specimens. Lavage fluid was centrifuged at 80,000 rpm for six minutes and smears were prepared from the sediment. BALC routinely included Papanicolaou and rapid modified Gomori's methenamine silver staining of ethanol-fixed smears. All of the 183 BAL specimens were considered adequate for cytologic evaluation as evidenced by greater than ten alveolar macrophages per high power field and no significant amounts of mucus or excessive numbers of epithelial cells. The TBB tissues were fonnalin-fixed and paraffin embedded. Sections of these biopsy specimens were evaluated with hematoxylin-eosin, modified Gomori's methenamine silver, and Kinyoun's acid-fast stains. Eleven of the 148 available biopsy specimens were considered diagnostically inadequate due to insufficient or absent pulmonary alveolar tissue. FOr the purposes of the study, diagnostically inadequate biopsy specimens were considered to be negative. For the purposes of the study, a given specimen was considered positive for an infectious agent if detected by any one of the three different procedures (BALC, TBB, culture) evaluated.

REsuLTS Forty-two bronchoscopies, from 36 patients, yielded no infectious organisms. Of all the bronchoscopy procedures, 141 (77 percent) from 95 patients were positive for one or more infectious agents; 76 of these specimens (53.9 percent of the total positive specimens) yielded a single pathogen; 55 of these specimens (39 percent of the total positive specimens) yielded two pathogens; in ten cases, three different infectious agents were identified. A summary of the significant pathogens isolated is tabulated in Table 1. A more Table 2-Comparome ~ ofBALC and TBBfor Dmction ofPneumocystis carinii

BALC TBB

No. Positive/fatal Positive Cases

Sensitivity

Predictive Value of Negative Test

9CW2 01180•

97.8% 83.6%

97.9% 84%

•Denominator differs because 12 of the 92 cases did not yield biopsy specimens.

Table 3-Comparome ~of BALC, TBB, and Culture for Dmction of Cytomegalovirus No. Positive/fotal Positive Cases BALC TBB BALCorTBB Culture

UW1 6179• 11191 89t~H

Sensitivity 11.0% 7.6% 12.1% 97.8%

•Denominator differs because 12 of the 91 cases did not yield biopsy specimens.

detailed analysis of several specific infectious agents follows. Pneumocystis carinii Pneumocystis carinii (Thble 2) was the most commonly identified infectious agent and was the most common pathogen diagnosed by both TBB and BALC. We found BALC to be more sensitive than TBB in detecting PC (97.8 percent vs 83.6 percent). The negative predictive value of BALC for diagnosis of PC was similarly better than that seen with TBB (97.9 percent vs 84 percent). Cytomegalovirus and Other Viruses

Cytomegalovirus (Table 3) was the second most common organism identified in the study material. Evidence of CMV was identified in nearly half (91 of 183) of all specimens and was present in 65 percent (91 of 141) of those specimens yielding significant pathogens. The overwhelming majority (80 of 91) of CMV-positive cases were detected by viral culture only. BALC and TBB histology demonstrated diagnostic nuclear and/or cytoplasmic inclusions in only a small percentage (11 percent and 7.6 percent, respectively) of those culture-positive cases for which comparison was possible. Characteristic CMV inclusion bodies were identified in one case each by BALC and TBB in which concurrent viral cultures were negative. Herpes simplex virus was isolated from three specimens by viral culture only. No morphologic changes indicative of HSV infection were identified by BALC or TBB in any of the material examined. Table 4-Comparome ~ ofBALC, TBB, and Culture for Dmction of Mycobacteria No. Positive/fotal Positive Cases BALC TBB BALCorTBB Culture

3126 5123•

1/'JR, 25126

Sensitivity 11.5% 21.7% 27% 96.1%

•Denominator differs because three of the 26 cases did not yield biopsy specimens. CHEST I 98 I 1 I JULY, 1990

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Table 5-Combined Senaitivity ofBALC and Culture for Detection o/Pneumocystis carinii, Cytomegalocirus, and Mycobacteria

PC CMV MB All pathogens

No. Positive/Total Positive Cases

Sensitivity

90192 90191 25126 2121216

97.8% 98.9% 96.1% 98.1%

Mycobacteria Culture demonstrated MB (Table 4) in 25 of the bronchoscopy specimens (17 isolates of M aviumintrocellulare, four isolates each of M kansasii, and M tuberculosis. Most of the mycobacteria-positive specimens (19 of 26) were detected by culture only. Both TBB and BALC were insensitive in detecting acid-fast organisms (sensitivities of 21.7 and 11 .5 percent, respectively). Each of the two cases detected by both BALC and culture yielded MK. Except for the presence of acid-fast bacilli, BALC in these patients demonstrated no particular cytologic changes which would have enabled a distinction from MB-negative specimens. Only one of the five TBB-positive cases showed a granulomatous tissue response in addition to the presence of acid-fast bacilli; this was the only case for which TBB, BALC, and culture were all positive for MB: MTB was isolated from this specimen. The BALC from this case showed no cytologic evidence of granulomas or any other distinctive changes. The other four MB-positive TBB showed only acidfast bacilli within interstitial tissues, without granulomatous inflammation: MAl was recovered from three of these specimens. In the fourth, a single acid-fast bacillus was identified by TBB: concurrent culture and BALC were both negative. Fungi

Cryptococcus sp organisms were detected, by culture only, in three specimens: in none of these cases was yeast detected by either BALC or TBB. One case of coccidiodomycosis was detected by both culture and BALC: TBB was not performed on this patient. Candida sp organisms were a very common finding in the study material. Twenty-two of 183 BALCs and 40 of 180 fungal cultures yielded Candida sp organisms. In none of these cases for which comparative material was available did the concurrent TBB show any evidence of invasive candidiasis. DISCUSSION

Several previous studiess- 10·13 ·14 have highlighted the utility of fiberoptic bronchoscopy as a diagnostic tool in the evaluation of AIDS-related opportunistic infections of the lung. These studies have demonstrated 26

that, for the detection of pulmonary pathogens, the sensitivity, specificity, and diagnostic yield of tests performed on bronchoscopically obtained specimens are consistently and reliably high. Given this knowledge, and the recognition of a high risk:benefit ratio for performance of open-lung biopsies on debilitated immunocompromised hosts, 15 many institutions (ours included) emphasize bronchoscopy as the primary diagnostic modality for the work-up of pulmonary infection in AIDS patients. While agreeing with the overall efficacy of bronchoscopy, of specific interest to us in the present study were the relative sensitivities of the three most commonly performed bronchoscopic procedures: BALC, TBB, and culture. The large number of specimens reviewed in this investigation provided a good picture of the diagnostic usefulness of these three procedures. The most prevalent cause of life-threatening opportunistic pulmonary infection among individuals with AIDS,4.1 6 • 17 and the most common pathogen identified in our study material was PC. Under most circumstances, fiberoptic bronchoscopy is considered the procedure of choice for the diagnosis of this infection.1&.18 The sensitivity of bronchoscopy has been reported by others to approach 100 percent when TBB and BALC are used in concert, 5 · 6 and some investigators 13 ·19 have advocated BALC as the exclusive diagnostic modality for PC. In our hands, BALC was more sensitive than TBB (97 .8 vs 83.6 percent), with BALC alone detecting 90 of the 92 cases. In the 80 cases for which comparative material was available, TBB was negative or inadequate in 13 cases. These results suggest that BALC without TBB is an adequate alternative to the performance of both procedures for the diagnosis of PC pneumonia in AIDS . The second most frequent infectious agent identified in this study was CMV. Although morphologic evidence for CMV was uncommon (only 12.1 percent of cases; see Table 3) nearly half of all the bronchoscopies (89 of the 180 which were cultured) grew CMV in culture. Fully 80 of the 91 (88 percent) CMVpositive bronchoscopies yielded virus by culture only. Other investigators9 •13·20 have noted the poor sensitivities ofboth TBB and BALC for detection of pulmonary CMV in AIDS patients, when compared to simultaneously performed viral culture studies. The clinical significance of this discrepancy remains unclear, however, as there is no consensus as to what actually constitutes a diagnosis of CMV pneumonia. At least one recent study21 suggests that recovery of CMV from the lungs of AIDS patients has no pathogenic significance per se. Most investigators would agree that those cases in which viral culture represents the only evidence of CMV pulmonary infection are problematic at best. We believe, as do others, 18·22 •23 that criteria in addition to positive culture results are Bronchoscopy Specimens in Adults with AIDS (WB/don-Unne, Rhone, Bouressa)

generally required to make a diagnosis of CMV pneumonia. Nevertheless, a positive CMV culture, in the absence of other clinical or bronchoscopic findings, may indicate a patient who needs further evaluation. Whatever the case, in our hands TBB added little to the diagnostic work-up of CMV pneumonitis. Eightynine of the 91 cases with evidence of CMV were positive by viral culture, and ten of the 11 cases demonstrating morphologic features of CMV were detected by BALC. In only one patient was TBB the sole evidence of CMV infection. The significance of the three HSV isolates in this study is unclear. In none of these three cases did BALC or TBB demonstrate diagnostic viral inclusions and none of these patients were thought clinically to have HSV pneumonitis. We presume the positive cultures to have been the result of upper airway contamination. Our experience suggests that recovery of HSV in viral cultures from AIDS-related bronchoscopy material should be interpreted with caution. Twenty-five of our bronchoscopy specimens yielded mycobacterial organisms by culture (17 cases of MAl, four cases each of MK and MTB). Neither BALC nor TBB were of any particular benefit in the diagnosis of MB-related disease. Morphologic evidence ofMB was seen in only seven cases (two by BALC only, four by TBB only, one by both TBB and BALC): in only one of these cases (which yielded MTB by culture) was a granulomatous tissue response identified by TBB. In those cases in which MB were detected by BALC, the identification was based solely on the presence of acid-fast bacilli. None of the three MB-positive BALCs (including the case in which TBB showed granulomas) displayed any cytologic evidence of granulomatous inflammation or other changes which would have enabled a distinction from MB-negative cases. As noted previously, TBB from one patient showed a single acid-fast bacillus concurrent with negative BALC and negative mycobacterial culture. The clinical significance of the TBB finding in this patient is unclear: he died within five months of bronchoscopy without ever manifesting other evidence of mycobacterial disease. Analogous to the situation already discussed concerning CMV, the clinical significance of recovery of nontuberculous MB from the lungs of AIDS patients remains uncertain. 6·16 One recent study24 concluded that positive bronchoscopic cultures for NTMB have no predictive value for disseminated NTMB disease in AIDS patients. The criteria for initiation of treatment for NTMB disease are poorly defined5 and therapy for this disease among AIDS patients has been largely unrewarding. 5 · 18 Nonetheless, as is the case with CMV, isolation of NTMB from bronchoscopy material probably warrants careful observation of the patient and may indicate a need for further workup. 10•24

Our experience with fungal pathogens in this study was limited. In none of the four cases (three isolates of Cryptococcus sp, one isolate of Coccidioides immitis) did BALC or TBB detect a pathogen that was not identified by culture. In fact, only one of the four cases was identified at all morphologically (by BALC in the case of coccidioidomycosis). Perhaps of greater significance was our observation that Candida sp organisms are identified with great frequency in bronchoscopy material from AIDS patients. Culture and/or BALC yielded Candida sp yeast in 40 of the 183 specimens evaluated. In none of these cases, for which comparative material was available, did TBB show morphologic evidence of invasive candidiasis. Candida sp is a very uncommon pulmonary pathogen in AIDS patients. 17·25 Most such organisms recovered from bronchoscopy in AIDS patients probably represent contamination from oropharyngeal infection. The diagnosis of Candida sp pneumonia in these patients should not be made without biopsy evidence of invasive disease. Six of our cases showed TBB features either diagnostic or suggestive of Kaposi's sarcoma. Not surprisingly, BALC in these cases revealed no cytologic evidence of KS. While the diagnosis of KS was the only situation for which TBB provided any clear-cut diagnostic advantage over BALC, it should be pointed out that previous studies have proved TBB itself to be an insensitive tool for diagnosis of pulmonary KS .u. 26 If definitive diagnosis of KS of the lungs is required, open lung biopsy may be indicated. In reviewing our material, we found that TBB added little to the combined use of BALC and culture in the evaluation of AIDS-related lung disease with the exception, already noted, of KS. As indicated in Table 5, the combined sensitivity of BALC and culture for detection of major pathogens in this study was in excess of 98 percent. Given that TBB is responsible for most of the complications associated with bronchoscopy,3·17 our results support the exclusion ofTBB as part of the routine bronchoscopic workup of nonneoplastic pulmonary disease in AIDS patients. Indeed, had TBB been excluded from the protocol in this series, only 4 pathogens would have gone undetected: two cases of PC, and one case each of CMV and MB (the latter, as already noted, of questionable clinical significance). Certainly our findings justify reassurance of the clinician as to the sensitivity of bronchoscopy in those patients in whom TBB is contraindicated for clinical reasons (such as thrombocytopenia). In summary, we conclude that, in patients with AIDS, fiberoptic bronchoscopy is a sensitive and effective procedure for the evaluation of pulmonary infections, and TBB may not be necessary as a routine procedure, in the work-up of nonneoplastic pulmonary CHEST I 98 I 1 I JULY. 1990

27

disease, in cases where both BALC and culture are performed at bronchoscopy. This study demonstrates that TBB adds little to the combined sensitivity of BALC and lung cultures in AIDS patients and that it should only be performed with a clear recognition of its potential risks and benefits. REFERENCES 1 Centers for Disease Control. Pneumocystis pneumonia- Los Angeles. Morbid Mortal Week Rep 1981; 30:250-52 2 Centers for Disease Control. Kaposi's sarcoma and Pneumocystis pneumonia among homosexual men-New York City and California. Morbid Mortal Week Rep 1981; 30:3()5..()8 3 Hopewell PC, Luce JM. Pulmonary involvement in the acquired immunodeficiency syndrome. Chest 1985; 87:104-12 4 Marchevsky A, Rosen MJ, Chrystal G, Kleinennan J. Pulmonary complications of the acquired immunodeficiency syndrome: a clinicopathologic study of70 cases. Hum Pathol1985; 16:659-70 5 Murray JF, Felton CP, Garay SM, Gottlieb MS, Hopewell PC, Stover DE, et al. Pulmonary complications of the acquired immunodeficiency syndrome: report of a National Heart, Lung, and Blood Institute workshop. N Eng! J Med 1984; 310:1682-88 6 Broaddus C, Dalce MD, Stulbarg MS, Blumenfeld W. Hadley K, Golden JA, et al. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann Intern Med 1985; 102:74752 7 Duggan MA, Pomponi C, Robboy SJ. Pulmonary cytology of the acquired immune deficiency syndrome: an analysis of 36 cases. Diagn Cytopathol1986; 2:181-86 8 Francis ND, Goldin RD, FOrster SM, Cook HT, Coleman DV, Shaw R, et al. Diagnosis of lung disease in acquired immune deficiency syndrome: biopsy or cytology and implications for management. J Clin Pathol 1987; 40:1269-73 9 Gal AA, Klatt EC, Koss MN, Strigle SM, Boylen Cl'. The effectiveness of bronchoscopy in the diagnosis of Pneumocystis carlnii and cytomegalovirus pulmonary infections in acquired immunodeficiency syndrome. Arch Pathol Lab Med 1987; 111:238-41 10 Stover DE, White DA, Romano PA, Gellene RA. Diagnosis of pulmonary disease in acquired immune deficiency syndrome (AIDS): role of bronchoscopy and bronchoalveolar lavage. Am Rev Respir Dis 1984; 130:659-62 11 Centers for Disease Control. Revision of the CDC surveillance case definition for acquired immunodeficiency syndrome. Morbid Mortal Week Rep 1987; 36 (suppi1S):1-15

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12 Centers for Disease Control. Human immunodeficiency virus infection in the United States: a review of current knowledge. Morbid Mortal Week Rep 1987; 36 (suppl S-6):1-48 13 DeFine LA, Saleba KP, Gibson BB, Wesseler TA, Baughman R. Cytologic evaluation of bronchoalveolar lavage specimens in immunosuppressed patients with suspected opportunistic infections. Acta Cytol1987; 31:235-42 14 Hartman B, Koss M, Hui A, Baumann W. Athos L, Boylen T. Pneumocystis carlnii pneumonia in the acquired immunodeficiency syndrome (AIDS): diagnosis with bronchial brushings, biopsy, and bronchoalveolar lavage. Chest 1985; 87:603-07 15 Tenholder MF. Pulmonary infections in the immunocompromised host: perspectives on procedures. Chest 1988; 94:676-78 16 Murray JF, Garay SM, Hopewell PC, Mills J, Snider GL, Stover DE. Pulmonary complications of the acquired immunodeficiency syndrome: an update. Am Rev Respir Dis 1987; 135:50409 17 Rankin JA, Collman R, Daniele RP. Acquired immune deficiency syndrome and the lung. Chest 1988; 94:155-64 18 Glatt AE, Chirgwin K, Landesman SH. Current concepts: treatment of infections associated with human immunodeficiency virus. N Eng! J Med 1988; 318:1439-48 19 Golden JA, Hollander H, Stulbarg MS, Gamsu G. Bronchoalveolar lavage as the exclusive diagnostic modality for Pneumocystis carlnii pneumonia: a prospective study among patients with acquired immunodeficiency syndrome. Chest 1986; 90:18-

22 20 Selvaggi SM , Gerber M. Pulmonary cytology in patients with the acquired immunodeficiency syndrome (AIDS). Diag Cytopathol1986; 2:187-93 21 Brodie HR, Broaddus C, Hopewell PC, Moss A, Mills J. Is cytomegalovirus (CMV) a cause of lung disease in patients with AIDS? Am Rev Respir Dis 1985; 131:227 22 Jacobson MA, Mills J. Cytomegalovirus infection. Clin Chest Med 1988; 9:443-48 23 Wallace JM, Hannah J. Cytomegalovirus pneumonitis in patients with AIDS: findings in an autopsy series. Chest 1987; 92:198203 24 Tenholder MF, Moser RJ, Tellis CJ. Mycobacteria other than tuberculosis: pulmonary involvement in patients with acquired immunodeficiency syndrome. Arch Intern Med 1988; 148:95355 25 Fels AOS. Bacterial and fungal pneumonias. Clin Chest Med 1988; 9:449-57 26 Garay SM, Belenko M, Fazzini E, Schinella R. Pulmonary manifestations of Kaposi's sarcoma. Chest 1987; 91:39-43

Brouchoscopy Specimens in Adulls with AIDS (Weldon-Unne, Rhone, BouruM)