Cytomegalovirus in the Lungs of Patients with AIDS Respiratory Pathogen or Passenger?1.2
ANN B. MILLAR, GARY PATOU, ROBERT F. MILLER, JANE E. GRUNDY, DAVID R. KATZ, IAN v. WELLER,3 and STEPHEN J. SEMPLE
Introduction Cytomegalovirus (CMV), a member of the herpesvirus family, commonly infects but rarely causes symptomatic disease in healthy individuals. Subsequently, latent infection is established during which the virus or viral genome resides intracellularly without replication but which may be reactivated by as yet poorly understood mechanisms. However, certain groups are at risk of symptomatic primary infection, such as neonates, or recurrent infection, such as the immunosuppressed, in particular recipients of renal or allogeneic bone marrow (2-4) transplants. In the latter patients the most serious complication of CMV infection is pneumonitis, which is associated with a mortality of up to 85070 (3). It might be anticipated that CMV would cause similar problems in patients with the acquired immune deficiency syndrome (AIDS) (1); indeed, CMV has been reported as present in the lungs of 17070 (5) to 19% (6) of these patients with respiratory problems. However, preliminary observations suggested that CMV in the lungs of AIDS patients was not associated with a poor clinical outcome (7-9). We therefore studied episodes of pneumonitis in these patients to determine the role of CMV as a lung pathogen in this group. Methods A total of 166sets of specimens from 144patients were studied, which came from 149patients who presented on 171 consecutive occasions with respiratory symptoms suggestive of pneumonitis. A minimum interval of 3 months and interim resolution of symptoms were required for separate episodes to be diagnosed in an individual patient. Three patients were too unwell to undergo fiberoptic bronchoscopy, and two refused this procedure. All the patients had AIDS (1) or were human immune deficiencyvirus (HIV) antibody positive. Of patients who were tested, 92010 had antibodies to CMV (Scan-Becton, Dickenson). No diagnosis had been made on conventional sputum culture (sputum was not in1474
SUMMARY A total of 166 consecutive clinical episodes of pneumonitis In patients with acquired Immune deficiency syndrome (AIDS) (1) or antibody positive for human Immune deficiency virus (HIV) were investigated for evidence of cytomegalovirus (CMV)Infection In their lungs and at peripheral sites to determine the pathogenicity of this virus in the lung and its relationship to peripheral CMV shedding. Evidence of CMVinfection was sought in bronchoalveolar lavage (BAL) fluid, blood, saliva, and urine using a specific monoclonal antibody to antigens produced by CMV-Infectedcells within 24 h. Although CMV was detected in 31 (19%) of BAL fluid specimens, In only six episodes was this the sole pathologic finding. In the remaining episodes either another infectious agent, Kaposi's sarcoma, or lymphoid Interstitial pneumonitis was found or no pathogen was detected. None of the patients were given specific antl-CMV treatment, and all but two recovered, including those patients In whom CMV was the sole finding at BAL. The presence of peripheral shedding of CMV did not have any significance in mortality or morbidity. Our findings are In direct contrast to those In recipients of allogeneic bone marrow transplants, In whom CMV pneumonitis is associated with a high mortality. We postulate that this difference is because AIDS patients cannot mount the destructive Immune response to CMV in the lung, which we believe to be the basis of the pathology seen in the former group. We conclude that CMV Is not a pathogen in the lungs of patients with HIV Infection, and we suggest that Its presence at this site does not warrant specific therapy In these patients. However, If current or future therapies for HIV restore the Immune capabilities of AIDS patients, we predict that life-threatening CMV pneumonitis may develop. AM REV RESPIR DIS 1990; 141:1474-14n
duced by saline inhalation) or cytologic sputum examination. Bronchoscopy was performed on each patient. The bronchoscope was passed into the lung and abutted into a subsegmental bronchus. Unless a focal abnormality was seen on the chest radiograph, lavage was performed in the lateral segment of the right lower lobe. Aliquots (60 ml) of isotonic buffered saline at 37° C were instilled via the bronchoscope and then aspirated to a total of 100ml aspirate or a maximum of 360ml instillate. Transbronchial biopsy was then performed. All specimens were transported to the appropriate laboratories on the day they were taken and in the majority within 3 h of removal from the patient. Bacteriologic investigation was performed on bronchoalveolar lavage (BAL) fluid and transbronchial biopsies and included Gram and Ziehl-Neelsen staining and culture for bacteria, mycobacteria, and fungi. The transbronchial biopsies and BAL fluid also underwent histologic and cytologic examination. Cytologic examination routinely includes assessment for intranuclear or intracytoplasmic viral changes in sputum and BAL fluid from these HI V-positive patients. Hemotoxylin and eosin stains and Grocott's silverstain wererou-
tinely used on the biopsies and Grocott's silver stain and Giemsa stain on the BAL fluid. Further staining procedures were performed as required on the basis of the preliminary findings. The biopsies wereretrospectivelyexamined specifically for evidence of pneumonitis (thickened alveolar septi, intra-alveolar exudate, and the presence of inflammatory cells)and the presence of viral inclusion bodies by an independent pathologist (Katz) who was unaware of the patients' clinical details. Specimensof saliva(in viral transport medium), urine, and blood (in preservative-free heparin) were taken from each patient at the
(Received in original form May 4, 1989 and in revised form September 27, 1989) I From the Departments of Medicine, Virology, Pathology, and Genitourinary Medicine, University College and Middlesex School of Medicine, and the Department of Virology, Royal Free Hospital, London, England. 2 Correspondence and requests for reprints should be addressed to Dr. Ann Millar, Department of Medicine, University College and Middlesex School of Medicine, Middlesex Hospital, Mortimer Street, London WIN 8AA, England. 3 Supported by the Wellcome Trust.
1475
CMV IN THE WNGS OF PATIENTS WITH AIDS
TABLE 1 ANALYSIS OF PATIENT DATA IN THOSE WITH CMV-POSITIVE BAL FLUID CMV Shedding Pt Episode 1a
1b
Saliva
Urine
Blood
+ +
+ +
NT NT Toxic Toxic
2a
2b 3 4 5 6 7 8 9 10
+
+
11
+
Toxic
Other Pathologic Findings PC PC PC S. pneumoniae + viral inclusions PC PC recurrence PC PC PC Negative PC H. influenzae + S. typhimurium PC PC PC PC PC PC + H. influenzae P. aeruginosa PC Negative Viral inclusions Negative PC PC PC + viral inclusions PC Negative S. aureus Negative PC
+ +
+
Toxic
+ +
Toxic
Toxic
NT
12 13 14 15 16a
16b 17a
17b
+ NT Toxic
+ + + +
+ + + + +
He 18a
18b
Toxic
+
+
Toxic r
19
20 21
+ +
22
NT
+ NT NT NT Toxic NT Toxic
+
+
Toxic Toxic
NT
23 24
25
+
Definition of abbreviations: PC
= Pneumocystis carin;;;
NT
= not tested; A
time of bronchoscopy. The presence of CMV was sought by the detection of early antigen fluorescent foci (DEAFF test) in inoculated cell cultures using CMV-specific monoclonal antibodies, which gives a result within 24 h (10). This technique is well established, and excellent sensitivity and specificity have been reported in comparison to other techniques, including conventional cell culture and direct monoclonal antibody assay on BAL cell sediment (11, 12). Fisher's exact test was used to compare mortality rates between groups (13). The relative risk (and 95070 confidence intervals) for mortality of all patients, those with Pneumocystis carinii pneumonia (PCP), with and without concomitant CMV, and those with and without peripheral CMV shedding are given (14).
Results
A positive diagnosis was made in 146 of the 166 respiratory episodes (81070) studied. Some patients presented on more than one occasion, and the figures quoted are individual clinical episodes with the figure in parentheses referring to the number of patients involved. BAL fluid was positive for CMV in 31 (25) of the 166(19070) episodes (table 1), and during
+ W
Outcome A+W A+W A+W A+W A+W Dead Dead A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W
SAL Cytology for CMV
+
+ +
+ + +
+
+
+
A+W A+W A+W A+W A+W A+W
+
= alive and well 2 months after presentation.
21 (20) of these episodes concomitant PCP was found. One of these 21 cases had in addition Hemophilus influenzae, and 19 (18) of these episodes resulted in recovery when treatment was given for PCP with standard therapy (cotrimoxazole, 120mg/kg/day). The remaining two (2) episodes resulted in death, one of which was a recurrence of PCP. In four (4) further episodes with CMV-positive lavage, coexisting bacterial pathogens (Streptococcus pneumoniae, H. influenzae and Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa) were found, and recovery occurred in all cases with appropriate antibiotic treatment. Thus during 25 (22) episodes, CMV was detected in BAL fluid in conjunction with a second pathogen. In six (5) episodes CMV alone was detected in BAL fluid, and these patients recovered on no specific anti-CMV therapy; two of them presented on a separate occasion with coexistent pathogens, and three of them were treated empirically with cotrimoxazole. Overall, only two deaths occurred following 31 (25) clinical episodes
with CMV-positiveBAL fluid, which represents a mortality rate of 6%. Of the 31 transbronchial biopsies from the episodes in which CMV was detected in BAL fluid, 30 were available for review. Nonspecific pneumonitis was seen in 21 specimens, inflammatory cells alone in four specimens, no alveolar tissue in three specimens, and no abnormality in two specimens. Both of the patients who died had histologic evidence of nonspecific pneumonitis but no histologic evidenceof CMV in their transbronchial biopsy. Three specimens showed cytopathic changes suggestive of CMV, and these came from two patients with copathogens (S. pneumoniae and P. carini/) in whom conventional therapy resulted in full recovery and one patient with this sole finding. Cytologic examination of BAL showed viral inclusions suggestive of CMV infection in 10episodes. The clinical presentations and outcome of the episodes during which CMV was not detected in BAL fluid are shown in table 2. In 80 (77) of these episodes, PCP alone was found (including one case in which viral inclusions were seen), of which seven resulted in death. One of these deaths was in a patient with a second episode of PCP. Tenof these patients represented with another respiratory diagnosis. Three (3) episodes of coexistent PCP and Mycobacterium tuberculosis, one of coexistent PCP and Mycobacterium avium intracellulare (MAl), and another of coexistent PCP and S. aureus, occurred; all resulted in recovery. Nine (8) episodes were attributed to intrapulmonary Kaposi's sarcoma (KS). Six (6) episodes of pneumococcal pneumonia were diagnosed and all six patients survived. In three (3) episodes the diagnosis of lymphoid interstitial pneumonitis was made. Four (4) episodes of S. aureus and another of coexistent S. aureus and M tuberculosis were diagnosed; all five resulted in recovery. One patient had actinomycosis and made a full recovery with antibiotic therapy. Examinations of 20 BAL fluids and transbronchial biopsies werenegative.All of these wereobtained from patients who remain aliveand well6 months after their presentation. Seven of these patients had cutaneous KS and radiographic features suggestiveof intrapulmonary KS.Another of these patients had a history and chest radiograph highly suggestive of PCP and despite his negative bronchoscopy responded to treatment with cotrimoxazole. Seven deaths occurred in 135(119) clin-
1476
MILLAR, PATOU, MILLER, GRUNDY, KATZ, WELLER, AND SEMPLE
TABLE 2
SAL
DIAGNOSIS AND OUTCOME OF CLINICAL EPISODES IN THOSE WITH CMV·NEGATIVE BAL FLUID
Episodes
79 1
3 1 1
9 1 6 4
2 1
2 1
1
20 3
Pathologic Findings PC PC + viral inclusions PC + AFB PC + MAl PC + S. aureus Kaposi's sarcoma Kaposi's sarcoma + MAl
S. pneumoniae S. aureus H. influenzae S. aureus + MTB MTB MAl Actinomycosis Negative LIP
Outcome
14(166) BAL Cytology for CMV
7 Died (1 was second episode, remainder A + W) A + W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W
+
12(164)
5(123)
Saliva
Urine
Fig. 1. Venn diagram showing the relative incidence of specimens for CMV. In each circle the number in parentheses refers to the total number of specimens in category. There were th ree positive blood specimens not shown in the figure.
Definition of abbreviations: PC = Pneumocystis carinii; AFB = acid-fast bacillus; MTB = Mycobacterium tuberculosis; MAl = Mycobacterium avlum intracellulare; LIP .. lymphoid interstitial pneumonitis; A + W alive and well 2 months after presentation.,
=
ical episodes when CMV was not detected in BAL fluid (table 2), which represents a mortality of 5010 (table 3). If only the 115 (101) episodes in which BAL was negative for CMV - but in which there was another pathologic finding - are considered, then the mortality is 5070 (table 3). These mortality rates are not significantly different from those for clinical episodes with BAL positive for CMV when compared overall (p = NS), and the relative risk of death in patients with CMV-positive lavage was 1.24(95010 confidence interval, 0.27 to 5.7). In those episodes where PCP alone was detected, the relative mortality was also not significantly different (p = NS), and the relative risk of death in those with CMV-positive lavage was 1.18 (95010 confidence interval, 0.26 to 5.29). Of 135 transbronchial biopsies, 93 were available for review from the CMV-negative BAL group, and 71 showed evidence of nonspecific pneumonitis; inflammatory cells alone were seen in 8 specimens, but no abnormality was seen in 11 specimens. No alveolar tissue was seen in the remaining two specimens. In addition, cytopathic changes suggestive of CMV were seen in one specimen from a patient in whom PCP was found and in whom full recovery resulted from treatment with cotrimoxazole. The incidence of peripheral shedding in urine and saliva and its overall relationship to the findings in BAL fluid are shown in figure 1. Three positive blood specimens wereobtained. Peripheral shedding was detected in the absence and presence of CMV in BAL but with no differ-
ence in the death rate between the comparable groups shown in table 4 (p = NS) and a relative risk of death in BAL CMVnegative group of 0.86 (confidence interval 0.11 to 6.8) in those with peripheral CMV shedding. Discussion In only 31 (25) of 166(19010) episodes of pneumonitis in HIV-positive or AIDS patients was CMV found in the lungs in this study, despite the fact that 92010 of these patients were also CMV antibody positive and therefore at risk of reactivation. One explanation for this is that cases of CMV were missed using the DEAFF technique alone, but the prevalence reported is comparable with other studies that used a variety of diagnostic criteria
(5, 6). If the CMV detected in the present episodes were a pathogen, then as no antiCMV treatment was instituted, a greatly increased mortality would be expected.
None of these episodes were treated with specific anti-CMV therapy, and yet 29 (22) resulted in recovery. Both the episodes resulting in death were in patients being treated for coexistent PCP, one of whom had a recurrence of this illness, which is known to have a poor prognosis (5). In fact we found that the overall mortality of the BAL CMV-positive group was 6OJo (2 of 31) and that of the CMVnegative group was 5070 (7 of 135). It has been suggested that in AIDS patients direct replication of CMV may cause lung damage and that a direct immunosuppressive effect by CMV itself may occur (15). If this were the case we might expect a poor outcome in those patients with concurrent infection with CMV. If we only consider those episodes in patients with PCP, then the mortality between patients with PCP alone and PCP and CMV was comparable (9 versus 10%). Furthermore, in three episodes, one of which had CMV-negative BAL fluid, histologic evidence suggestive of CMV was seen, in two episodes copathogens were detected
TABLE 3 RELATIONSHIP BETWEEN CLINICAL OUTCOME AND THE PRESENCE OF CMV IN BAL ALONE AND WITH OTHER PATHOLOGIC FINDINGS
Episodes CMValone CMV + PC CMV + other pathology Total PC alone Other pathologic findings atone No pathology detected Total
Deaths
Percentage of Total
6
o
o
21 4
2
10
o
o
31
2
6
80 35
7
9
20
o o
o
135
7
5
Definition of abbreviation: PC = Pneumocystis carinil.
o
14n
CMV IN THE WNGS OF PATIENTS WITH AIDS
TABLE 4 RELATIONSHIP BETWEEN PERIPHERAL SHEDDING OF CMV, ITS PRESENCE IN BAL flUID, AND OUTCOME CMV BAL
+ + +
CMV Peripheral (patients)
Number of Episodes
+ + Total
20 (15) 11 (10)
~
31 (25)
2 (6%)
22 (20) 113 (101)
+ Total
Deaths
135 (119)
2
1
~ 7 (5%)
carinii and S. pneumoniae), but recovery occurred in all without specific antiCMV treatment. Weconclude that CMV is not frequently found in the lungs of patients with or at risk of HIV infection and that its presence does not imply a pathogenic role at this site, as treatment of copathogens leads to clinical recovery in most cases. This apparent lack of pathogenic effect of CMV in the AIDS patients is in marked contrast to the situation in allogeneic transplant recipients (2, 16), in whom the mortality approaches 85070 (2). It is of interest that in allogeneic bone marrow transplant recipients, graft-versushost disease is a risk factor for the development of CMV pneumonitis (17). Furthermore, the antiviral agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine, which has been successfully used to treat CMV retinitis (18, 19), a condition presumed to be due to direct viral replication and damage (20), has been less successful in the treatment of CMV pneumonitis in transplant recipients when used as sole therapy (18), although more successful when used in combination with gammaglobulin (21,22). These observations have led to the proposal that CMV'pneumonitis in transplant recipients is an immunopathologic condition due to a T cell response to a virally induced antigen expressed on cells in the lung (23) and that variations in the immune reactivity associated with graft-versus-host disease are related to pathogenesis. Although it is not known whether the cytotoxic T cell, helper T cell, or hypersensitivity T cell subset is primarily involved, data from a murine mouse model favored the latter (23). Since AIDS patients have a deficiency of CD4-positive T cells, which include (R
the helper and delayed hypersensitivity subsets, we suggest that although CMV is present in their lungs, they lack the necessary destructive immune response that we believe leads to CMV pneumonitis in allogeneic transplant recipients. Any additional role of CD8-positive T cells would also be diminished in AIDS patients since their cytotoxic activity is reduced (24). It is clear from the results presented here that the detection of CMV in BAL fluid or the presenceof histologicchanges suggestiveof CMV in lung biopsies from AIDS patients does not establish this virus as the primary cause of pneumonitis. In seven cases in whom CMV was the only potential pathogen detected, no specific anti-CMV therapy was given and recovery ensued, which suggests that CMV is acting as a passenger, not a pathogen. However, we must bear in mind for the future that if treatment of AIDS patients for their HIV infection results in reconstitution of their immune responses, CMV may become a pathogen in their lungs as is currently the case in transplant recipients. Acknowledgment The writers thank Annette Skinner and Kathryn Young for their fortitude in the preparation of this manuscript, Brian Newman for his expert statistical guidance, and the pathologic services of the Middlesex Hospital. References 1. Update on acquired immune deficiency syndrome (AIDS). MMWR 1982; 31:507-14. 2. Fiala M, Payne JE, Berne TV, et 0/. Epidemiology of cytomegalovirus infection after transplantation and immunosuppression. J Infect Dis 1975; 132:421-33. 3. Meyers JD, Fownoy N, Thomas E. Risk factors for cytomegalovirus infection after human marrow transplantation. J Infect Dis 1986; 153:478-88. 4. Rubin RH, Tolkoff-Rubin NE, Oliver 0, et 0/. Multicentre seroepidemiologic study of the impact of cytomegalovirus infection on renal transplantation. Transplantation 1985; 40:243-9. 5. Murray JF, Felton CP, Garay SM, et 0/. Pulmonary complications of the acquired immune deficiency syndrome. Report of a National Heart, Lung, and Blood Institute workshop. N Engl J Med 1984; 310:1682-8. 6. Stover DE, White DE, Romano PA, Gellere 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. 7. 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 1987; 131(Suppl:A227). 8. Vittecoq 0, Durand S, Mazeron MC, Hirsch A, Perol Y. Is cytomegalovirus (CMV) a cause of pneumonia in AIDS? In: Abstracts of the Third International Conference on AIDS, Washington, DC, June 1-5,1987. Washington, DC: Department of Health and Human Services, 1987; 86. 9. Gardner TO, Youle M, Hanson PJV, Griffiths PO, Collins JV, Gazzard BG. J Infect 1989; 18:11-117. 10. Griffiths PO, Panjwani DO, Stirk PR, et 0/. Rapid diagnosis of cytomegalovirus infection in immuno-compromised patients by detection of early antigen fluorescent foci. Lancet 1984;2:1242-468. 11. Stirk PR, Griffiths PD. Use of monoclonal antibodies for the detection of cytomegalovirus infection by the detection of early antigen fluorescent foci (DEAFF) in cell culture. J Med Viro11987; 21:329-37. 12. Stirk PR, Griffiths PD. Comparative sensitivity of three methods for the diagnosis of cytomegalovirus lung infection. 1 Virol Methods 1988; 20:133-42. 13. Fisher RA. Statistical methods for research workers. 14thed. Edinburgh: Oliver and Boyd, 1970. 14. Morris JA, Gardner MJ. Calculating confidence intervals for relative risks (odds ratios) and standardised ratios and rates. Br Med J 1988; 296: 1313-6. 15. Wallace JM. Pulmonary infection in human immunodeficiency disease: viral pulmonary infections. Semin Respir Med 1989; 4:147-54. 16. Wingard JR, Mellits ED, Sostrin MB. Interstitial pneumonitis after allogeneic bone marrow transplantation. Medicine (Baltimore) 1988;67:175-86. 17. Millar W, Flynn P, McCullough J, et 0/. Cytomegalovirus infection after bone marrow transplantation. An association with graft-vs-host disease. Blood 1986; 67:1162-7. 18. Collaborative DHPG Treatment Study Group. Treatment of serious cytomegalovirus infections with 9-(l,3-dihydroxy-2-propoxymethyl)guanine in patients with AIDS and other immunodeficiencies. N Engl J Med 1986; 314:801-5. 19. Felenstein 0, D'Amico OJ, Hirsch MS, et 0/. Treatment of cytomegalovirus retinitis with 9-[2hydroxyl-l-(hydroxymethyl) ethoxymethyljguanine, Ann Intern Med 1985; 103:377-83. 20. Griffiths PO, Grundy JE. The status of CMV as a human pathogen. Epidemiol Infect 1988; 100: 1-15. 21. Emanuel 0, Jules-Elysee K, Cummingham I, et 0/. Cytomegalovirus pneumonia after bonemarrow transplantation successfully treated with the combination of ganciclovir and high-dose intravenous immune globulin. Ann Intern Med 1988; 109:777-82. 22. Reed EC, Bowden RA, Dandlifer PS, Lilleby KE, MeyersJD. Treatment ofCMV pneumonia with gancicovir and intravenous immunoglobulin patients with bone marrow transplants. Ann Intern Med 1988; 109:783-8. 23. Grundy JE, Shanley 10, Griffiths PD. Is cytomegalovirus pneumonitis in transplant patients an immuno-pathological condition? Lancet 1988; 2:996-9. 24. Agnostini C, Poletti V, Zamballo R, et 0/. Phenotypical and functional analysis of bronchoalveolar lavage lymphocytes in patients with HIV infection. Am Rev Respir Dis 1989; 138:1609-15.
ANN B. MILLAR, GARY PATOU, ROBERT F. MILLER, JANE E. GRUNDY, DAVID R. KATZ, IAN v. WELLER,3 and STEPHEN J. SEMPLE
Introduction Cytomegalovirus (CMV), a member of the herpesvirus family, commonly infects but rarely causes symptomatic disease in healthy individuals. Subsequently, latent infection is established during which the virus or viral genome resides intracellularly without replication but which may be reactivated by as yet poorly understood mechanisms. However, certain groups are at risk of symptomatic primary infection, such as neonates, or recurrent infection, such as the immunosuppressed, in particular recipients of renal or allogeneic bone marrow (2-4) transplants. In the latter patients the most serious complication of CMV infection is pneumonitis, which is associated with a mortality of up to 85070 (3). It might be anticipated that CMV would cause similar problems in patients with the acquired immune deficiency syndrome (AIDS) (1); indeed, CMV has been reported as present in the lungs of 17070 (5) to 19% (6) of these patients with respiratory problems. However, preliminary observations suggested that CMV in the lungs of AIDS patients was not associated with a poor clinical outcome (7-9). We therefore studied episodes of pneumonitis in these patients to determine the role of CMV as a lung pathogen in this group. Methods A total of 166sets of specimens from 144patients were studied, which came from 149patients who presented on 171 consecutive occasions with respiratory symptoms suggestive of pneumonitis. A minimum interval of 3 months and interim resolution of symptoms were required for separate episodes to be diagnosed in an individual patient. Three patients were too unwell to undergo fiberoptic bronchoscopy, and two refused this procedure. All the patients had AIDS (1) or were human immune deficiencyvirus (HIV) antibody positive. Of patients who were tested, 92010 had antibodies to CMV (Scan-Becton, Dickenson). No diagnosis had been made on conventional sputum culture (sputum was not in1474
SUMMARY A total of 166 consecutive clinical episodes of pneumonitis In patients with acquired Immune deficiency syndrome (AIDS) (1) or antibody positive for human Immune deficiency virus (HIV) were investigated for evidence of cytomegalovirus (CMV)Infection In their lungs and at peripheral sites to determine the pathogenicity of this virus in the lung and its relationship to peripheral CMV shedding. Evidence of CMVinfection was sought in bronchoalveolar lavage (BAL) fluid, blood, saliva, and urine using a specific monoclonal antibody to antigens produced by CMV-Infectedcells within 24 h. Although CMV was detected in 31 (19%) of BAL fluid specimens, In only six episodes was this the sole pathologic finding. In the remaining episodes either another infectious agent, Kaposi's sarcoma, or lymphoid Interstitial pneumonitis was found or no pathogen was detected. None of the patients were given specific antl-CMV treatment, and all but two recovered, including those patients In whom CMV was the sole finding at BAL. The presence of peripheral shedding of CMV did not have any significance in mortality or morbidity. Our findings are In direct contrast to those In recipients of allogeneic bone marrow transplants, In whom CMV pneumonitis is associated with a high mortality. We postulate that this difference is because AIDS patients cannot mount the destructive Immune response to CMV in the lung, which we believe to be the basis of the pathology seen in the former group. We conclude that CMV Is not a pathogen in the lungs of patients with HIV Infection, and we suggest that Its presence at this site does not warrant specific therapy In these patients. However, If current or future therapies for HIV restore the Immune capabilities of AIDS patients, we predict that life-threatening CMV pneumonitis may develop. AM REV RESPIR DIS 1990; 141:1474-14n
duced by saline inhalation) or cytologic sputum examination. Bronchoscopy was performed on each patient. The bronchoscope was passed into the lung and abutted into a subsegmental bronchus. Unless a focal abnormality was seen on the chest radiograph, lavage was performed in the lateral segment of the right lower lobe. Aliquots (60 ml) of isotonic buffered saline at 37° C were instilled via the bronchoscope and then aspirated to a total of 100ml aspirate or a maximum of 360ml instillate. Transbronchial biopsy was then performed. All specimens were transported to the appropriate laboratories on the day they were taken and in the majority within 3 h of removal from the patient. Bacteriologic investigation was performed on bronchoalveolar lavage (BAL) fluid and transbronchial biopsies and included Gram and Ziehl-Neelsen staining and culture for bacteria, mycobacteria, and fungi. The transbronchial biopsies and BAL fluid also underwent histologic and cytologic examination. Cytologic examination routinely includes assessment for intranuclear or intracytoplasmic viral changes in sputum and BAL fluid from these HI V-positive patients. Hemotoxylin and eosin stains and Grocott's silverstain wererou-
tinely used on the biopsies and Grocott's silver stain and Giemsa stain on the BAL fluid. Further staining procedures were performed as required on the basis of the preliminary findings. The biopsies wereretrospectivelyexamined specifically for evidence of pneumonitis (thickened alveolar septi, intra-alveolar exudate, and the presence of inflammatory cells)and the presence of viral inclusion bodies by an independent pathologist (Katz) who was unaware of the patients' clinical details. Specimensof saliva(in viral transport medium), urine, and blood (in preservative-free heparin) were taken from each patient at the
(Received in original form May 4, 1989 and in revised form September 27, 1989) I From the Departments of Medicine, Virology, Pathology, and Genitourinary Medicine, University College and Middlesex School of Medicine, and the Department of Virology, Royal Free Hospital, London, England. 2 Correspondence and requests for reprints should be addressed to Dr. Ann Millar, Department of Medicine, University College and Middlesex School of Medicine, Middlesex Hospital, Mortimer Street, London WIN 8AA, England. 3 Supported by the Wellcome Trust.
1475
CMV IN THE WNGS OF PATIENTS WITH AIDS
TABLE 1 ANALYSIS OF PATIENT DATA IN THOSE WITH CMV-POSITIVE BAL FLUID CMV Shedding Pt Episode 1a
1b
Saliva
Urine
Blood
+ +
+ +
NT NT Toxic Toxic
2a
2b 3 4 5 6 7 8 9 10
+
+
11
+
Toxic
Other Pathologic Findings PC PC PC S. pneumoniae + viral inclusions PC PC recurrence PC PC PC Negative PC H. influenzae + S. typhimurium PC PC PC PC PC PC + H. influenzae P. aeruginosa PC Negative Viral inclusions Negative PC PC PC + viral inclusions PC Negative S. aureus Negative PC
+ +
+
Toxic
+ +
Toxic
Toxic
NT
12 13 14 15 16a
16b 17a
17b
+ NT Toxic
+ + + +
+ + + + +
He 18a
18b
Toxic
+
+
Toxic r
19
20 21
+ +
22
NT
+ NT NT NT Toxic NT Toxic
+
+
Toxic Toxic
NT
23 24
25
+
Definition of abbreviations: PC
= Pneumocystis carin;;;
NT
= not tested; A
time of bronchoscopy. The presence of CMV was sought by the detection of early antigen fluorescent foci (DEAFF test) in inoculated cell cultures using CMV-specific monoclonal antibodies, which gives a result within 24 h (10). This technique is well established, and excellent sensitivity and specificity have been reported in comparison to other techniques, including conventional cell culture and direct monoclonal antibody assay on BAL cell sediment (11, 12). Fisher's exact test was used to compare mortality rates between groups (13). The relative risk (and 95070 confidence intervals) for mortality of all patients, those with Pneumocystis carinii pneumonia (PCP), with and without concomitant CMV, and those with and without peripheral CMV shedding are given (14).
Results
A positive diagnosis was made in 146 of the 166 respiratory episodes (81070) studied. Some patients presented on more than one occasion, and the figures quoted are individual clinical episodes with the figure in parentheses referring to the number of patients involved. BAL fluid was positive for CMV in 31 (25) of the 166(19070) episodes (table 1), and during
+ W
Outcome A+W A+W A+W A+W A+W Dead Dead A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W
SAL Cytology for CMV
+
+ +
+ + +
+
+
+
A+W A+W A+W A+W A+W A+W
+
= alive and well 2 months after presentation.
21 (20) of these episodes concomitant PCP was found. One of these 21 cases had in addition Hemophilus influenzae, and 19 (18) of these episodes resulted in recovery when treatment was given for PCP with standard therapy (cotrimoxazole, 120mg/kg/day). The remaining two (2) episodes resulted in death, one of which was a recurrence of PCP. In four (4) further episodes with CMV-positive lavage, coexisting bacterial pathogens (Streptococcus pneumoniae, H. influenzae and Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa) were found, and recovery occurred in all cases with appropriate antibiotic treatment. Thus during 25 (22) episodes, CMV was detected in BAL fluid in conjunction with a second pathogen. In six (5) episodes CMV alone was detected in BAL fluid, and these patients recovered on no specific anti-CMV therapy; two of them presented on a separate occasion with coexistent pathogens, and three of them were treated empirically with cotrimoxazole. Overall, only two deaths occurred following 31 (25) clinical episodes
with CMV-positiveBAL fluid, which represents a mortality rate of 6%. Of the 31 transbronchial biopsies from the episodes in which CMV was detected in BAL fluid, 30 were available for review. Nonspecific pneumonitis was seen in 21 specimens, inflammatory cells alone in four specimens, no alveolar tissue in three specimens, and no abnormality in two specimens. Both of the patients who died had histologic evidence of nonspecific pneumonitis but no histologic evidenceof CMV in their transbronchial biopsy. Three specimens showed cytopathic changes suggestive of CMV, and these came from two patients with copathogens (S. pneumoniae and P. carini/) in whom conventional therapy resulted in full recovery and one patient with this sole finding. Cytologic examination of BAL showed viral inclusions suggestive of CMV infection in 10episodes. The clinical presentations and outcome of the episodes during which CMV was not detected in BAL fluid are shown in table 2. In 80 (77) of these episodes, PCP alone was found (including one case in which viral inclusions were seen), of which seven resulted in death. One of these deaths was in a patient with a second episode of PCP. Tenof these patients represented with another respiratory diagnosis. Three (3) episodes of coexistent PCP and Mycobacterium tuberculosis, one of coexistent PCP and Mycobacterium avium intracellulare (MAl), and another of coexistent PCP and S. aureus, occurred; all resulted in recovery. Nine (8) episodes were attributed to intrapulmonary Kaposi's sarcoma (KS). Six (6) episodes of pneumococcal pneumonia were diagnosed and all six patients survived. In three (3) episodes the diagnosis of lymphoid interstitial pneumonitis was made. Four (4) episodes of S. aureus and another of coexistent S. aureus and M tuberculosis were diagnosed; all five resulted in recovery. One patient had actinomycosis and made a full recovery with antibiotic therapy. Examinations of 20 BAL fluids and transbronchial biopsies werenegative.All of these wereobtained from patients who remain aliveand well6 months after their presentation. Seven of these patients had cutaneous KS and radiographic features suggestiveof intrapulmonary KS.Another of these patients had a history and chest radiograph highly suggestive of PCP and despite his negative bronchoscopy responded to treatment with cotrimoxazole. Seven deaths occurred in 135(119) clin-
1476
MILLAR, PATOU, MILLER, GRUNDY, KATZ, WELLER, AND SEMPLE
TABLE 2
SAL
DIAGNOSIS AND OUTCOME OF CLINICAL EPISODES IN THOSE WITH CMV·NEGATIVE BAL FLUID
Episodes
79 1
3 1 1
9 1 6 4
2 1
2 1
1
20 3
Pathologic Findings PC PC + viral inclusions PC + AFB PC + MAl PC + S. aureus Kaposi's sarcoma Kaposi's sarcoma + MAl
S. pneumoniae S. aureus H. influenzae S. aureus + MTB MTB MAl Actinomycosis Negative LIP
Outcome
14(166) BAL Cytology for CMV
7 Died (1 was second episode, remainder A + W) A + W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W A+W
+
12(164)
5(123)
Saliva
Urine
Fig. 1. Venn diagram showing the relative incidence of specimens for CMV. In each circle the number in parentheses refers to the total number of specimens in category. There were th ree positive blood specimens not shown in the figure.
Definition of abbreviations: PC = Pneumocystis carinii; AFB = acid-fast bacillus; MTB = Mycobacterium tuberculosis; MAl = Mycobacterium avlum intracellulare; LIP .. lymphoid interstitial pneumonitis; A + W alive and well 2 months after presentation.,
=
ical episodes when CMV was not detected in BAL fluid (table 2), which represents a mortality of 5010 (table 3). If only the 115 (101) episodes in which BAL was negative for CMV - but in which there was another pathologic finding - are considered, then the mortality is 5070 (table 3). These mortality rates are not significantly different from those for clinical episodes with BAL positive for CMV when compared overall (p = NS), and the relative risk of death in patients with CMV-positive lavage was 1.24(95010 confidence interval, 0.27 to 5.7). In those episodes where PCP alone was detected, the relative mortality was also not significantly different (p = NS), and the relative risk of death in those with CMV-positive lavage was 1.18 (95010 confidence interval, 0.26 to 5.29). Of 135 transbronchial biopsies, 93 were available for review from the CMV-negative BAL group, and 71 showed evidence of nonspecific pneumonitis; inflammatory cells alone were seen in 8 specimens, but no abnormality was seen in 11 specimens. No alveolar tissue was seen in the remaining two specimens. In addition, cytopathic changes suggestive of CMV were seen in one specimen from a patient in whom PCP was found and in whom full recovery resulted from treatment with cotrimoxazole. The incidence of peripheral shedding in urine and saliva and its overall relationship to the findings in BAL fluid are shown in figure 1. Three positive blood specimens wereobtained. Peripheral shedding was detected in the absence and presence of CMV in BAL but with no differ-
ence in the death rate between the comparable groups shown in table 4 (p = NS) and a relative risk of death in BAL CMVnegative group of 0.86 (confidence interval 0.11 to 6.8) in those with peripheral CMV shedding. Discussion In only 31 (25) of 166(19010) episodes of pneumonitis in HIV-positive or AIDS patients was CMV found in the lungs in this study, despite the fact that 92010 of these patients were also CMV antibody positive and therefore at risk of reactivation. One explanation for this is that cases of CMV were missed using the DEAFF technique alone, but the prevalence reported is comparable with other studies that used a variety of diagnostic criteria
(5, 6). If the CMV detected in the present episodes were a pathogen, then as no antiCMV treatment was instituted, a greatly increased mortality would be expected.
None of these episodes were treated with specific anti-CMV therapy, and yet 29 (22) resulted in recovery. Both the episodes resulting in death were in patients being treated for coexistent PCP, one of whom had a recurrence of this illness, which is known to have a poor prognosis (5). In fact we found that the overall mortality of the BAL CMV-positive group was 6OJo (2 of 31) and that of the CMVnegative group was 5070 (7 of 135). It has been suggested that in AIDS patients direct replication of CMV may cause lung damage and that a direct immunosuppressive effect by CMV itself may occur (15). If this were the case we might expect a poor outcome in those patients with concurrent infection with CMV. If we only consider those episodes in patients with PCP, then the mortality between patients with PCP alone and PCP and CMV was comparable (9 versus 10%). Furthermore, in three episodes, one of which had CMV-negative BAL fluid, histologic evidence suggestive of CMV was seen, in two episodes copathogens were detected
TABLE 3 RELATIONSHIP BETWEEN CLINICAL OUTCOME AND THE PRESENCE OF CMV IN BAL ALONE AND WITH OTHER PATHOLOGIC FINDINGS
Episodes CMValone CMV + PC CMV + other pathology Total PC alone Other pathologic findings atone No pathology detected Total
Deaths
Percentage of Total
6
o
o
21 4
2
10
o
o
31
2
6
80 35
7
9
20
o o
o
135
7
5
Definition of abbreviation: PC = Pneumocystis carinil.
o
14n
CMV IN THE WNGS OF PATIENTS WITH AIDS
TABLE 4 RELATIONSHIP BETWEEN PERIPHERAL SHEDDING OF CMV, ITS PRESENCE IN BAL flUID, AND OUTCOME CMV BAL
+ + +
CMV Peripheral (patients)
Number of Episodes
+ + Total
20 (15) 11 (10)
~
31 (25)
2 (6%)
22 (20) 113 (101)
+ Total
Deaths
135 (119)
2
1
~ 7 (5%)
carinii and S. pneumoniae), but recovery occurred in all without specific antiCMV treatment. Weconclude that CMV is not frequently found in the lungs of patients with or at risk of HIV infection and that its presence does not imply a pathogenic role at this site, as treatment of copathogens leads to clinical recovery in most cases. This apparent lack of pathogenic effect of CMV in the AIDS patients is in marked contrast to the situation in allogeneic transplant recipients (2, 16), in whom the mortality approaches 85070 (2). It is of interest that in allogeneic bone marrow transplant recipients, graft-versushost disease is a risk factor for the development of CMV pneumonitis (17). Furthermore, the antiviral agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine, which has been successfully used to treat CMV retinitis (18, 19), a condition presumed to be due to direct viral replication and damage (20), has been less successful in the treatment of CMV pneumonitis in transplant recipients when used as sole therapy (18), although more successful when used in combination with gammaglobulin (21,22). These observations have led to the proposal that CMV'pneumonitis in transplant recipients is an immunopathologic condition due to a T cell response to a virally induced antigen expressed on cells in the lung (23) and that variations in the immune reactivity associated with graft-versus-host disease are related to pathogenesis. Although it is not known whether the cytotoxic T cell, helper T cell, or hypersensitivity T cell subset is primarily involved, data from a murine mouse model favored the latter (23). Since AIDS patients have a deficiency of CD4-positive T cells, which include (R
the helper and delayed hypersensitivity subsets, we suggest that although CMV is present in their lungs, they lack the necessary destructive immune response that we believe leads to CMV pneumonitis in allogeneic transplant recipients. Any additional role of CD8-positive T cells would also be diminished in AIDS patients since their cytotoxic activity is reduced (24). It is clear from the results presented here that the detection of CMV in BAL fluid or the presenceof histologicchanges suggestiveof CMV in lung biopsies from AIDS patients does not establish this virus as the primary cause of pneumonitis. In seven cases in whom CMV was the only potential pathogen detected, no specific anti-CMV therapy was given and recovery ensued, which suggests that CMV is acting as a passenger, not a pathogen. However, we must bear in mind for the future that if treatment of AIDS patients for their HIV infection results in reconstitution of their immune responses, CMV may become a pathogen in their lungs as is currently the case in transplant recipients. Acknowledgment The writers thank Annette Skinner and Kathryn Young for their fortitude in the preparation of this manuscript, Brian Newman for his expert statistical guidance, and the pathologic services of the Middlesex Hospital. References 1. Update on acquired immune deficiency syndrome (AIDS). MMWR 1982; 31:507-14. 2. Fiala M, Payne JE, Berne TV, et 0/. Epidemiology of cytomegalovirus infection after transplantation and immunosuppression. J Infect Dis 1975; 132:421-33. 3. Meyers JD, Fownoy N, Thomas E. Risk factors for cytomegalovirus infection after human marrow transplantation. J Infect Dis 1986; 153:478-88. 4. Rubin RH, Tolkoff-Rubin NE, Oliver 0, et 0/. Multicentre seroepidemiologic study of the impact of cytomegalovirus infection on renal transplantation. Transplantation 1985; 40:243-9. 5. Murray JF, Felton CP, Garay SM, et 0/. Pulmonary complications of the acquired immune deficiency syndrome. Report of a National Heart, Lung, and Blood Institute workshop. N Engl J Med 1984; 310:1682-8. 6. Stover DE, White DE, Romano PA, Gellere 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. 7. 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 1987; 131(Suppl:A227). 8. Vittecoq 0, Durand S, Mazeron MC, Hirsch A, Perol Y. Is cytomegalovirus (CMV) a cause of pneumonia in AIDS? In: Abstracts of the Third International Conference on AIDS, Washington, DC, June 1-5,1987. Washington, DC: Department of Health and Human Services, 1987; 86. 9. Gardner TO, Youle M, Hanson PJV, Griffiths PO, Collins JV, Gazzard BG. J Infect 1989; 18:11-117. 10. Griffiths PO, Panjwani DO, Stirk PR, et 0/. Rapid diagnosis of cytomegalovirus infection in immuno-compromised patients by detection of early antigen fluorescent foci. Lancet 1984;2:1242-468. 11. Stirk PR, Griffiths PD. Use of monoclonal antibodies for the detection of cytomegalovirus infection by the detection of early antigen fluorescent foci (DEAFF) in cell culture. J Med Viro11987; 21:329-37. 12. Stirk PR, Griffiths PD. Comparative sensitivity of three methods for the diagnosis of cytomegalovirus lung infection. 1 Virol Methods 1988; 20:133-42. 13. Fisher RA. Statistical methods for research workers. 14thed. Edinburgh: Oliver and Boyd, 1970. 14. Morris JA, Gardner MJ. Calculating confidence intervals for relative risks (odds ratios) and standardised ratios and rates. Br Med J 1988; 296: 1313-6. 15. Wallace JM. Pulmonary infection in human immunodeficiency disease: viral pulmonary infections. Semin Respir Med 1989; 4:147-54. 16. Wingard JR, Mellits ED, Sostrin MB. Interstitial pneumonitis after allogeneic bone marrow transplantation. Medicine (Baltimore) 1988;67:175-86. 17. Millar W, Flynn P, McCullough J, et 0/. Cytomegalovirus infection after bone marrow transplantation. An association with graft-vs-host disease. Blood 1986; 67:1162-7. 18. Collaborative DHPG Treatment Study Group. Treatment of serious cytomegalovirus infections with 9-(l,3-dihydroxy-2-propoxymethyl)guanine in patients with AIDS and other immunodeficiencies. N Engl J Med 1986; 314:801-5. 19. Felenstein 0, D'Amico OJ, Hirsch MS, et 0/. Treatment of cytomegalovirus retinitis with 9-[2hydroxyl-l-(hydroxymethyl) ethoxymethyljguanine, Ann Intern Med 1985; 103:377-83. 20. Griffiths PO, Grundy JE. The status of CMV as a human pathogen. Epidemiol Infect 1988; 100: 1-15. 21. Emanuel 0, Jules-Elysee K, Cummingham I, et 0/. Cytomegalovirus pneumonia after bonemarrow transplantation successfully treated with the combination of ganciclovir and high-dose intravenous immune globulin. Ann Intern Med 1988; 109:777-82. 22. Reed EC, Bowden RA, Dandlifer PS, Lilleby KE, MeyersJD. Treatment ofCMV pneumonia with gancicovir and intravenous immunoglobulin patients with bone marrow transplants. Ann Intern Med 1988; 109:783-8. 23. Grundy JE, Shanley 10, Griffiths PD. Is cytomegalovirus pneumonitis in transplant patients an immuno-pathological condition? Lancet 1988; 2:996-9. 24. Agnostini C, Poletti V, Zamballo R, et 0/. Phenotypical and functional analysis of bronchoalveolar lavage lymphocytes in patients with HIV infection. Am Rev Respir Dis 1989; 138:1609-15.
