The patient did well until June 1982, when he was hospital- ... kg of weight, and was readmitted to the hospital. ..... Children's Hospital Medical Center, Akron.
Acquired Immunodeficiency Syndrome with Pneumocystis carinii Pneumonia and Mycobacterium avium-intraceiiuiare Infection in a Previously Healthy Patient with Classic Hemophilia Clinical, Immunologic, and Virologic Findings MAN-CHIU POON, M.D.; ALAN LANDAY, Ph.D.; ED F. PRASTHOFER, M.D.; and SERGIO STAGNO, M.D.; Birmingham, Alabama A previously healthy patient with classic hemophilia who was on a home infusion program with factor VIII concentrates developed an acquired immunodeficiency syndrome'manifested by a dramatic weight loss ( 4 7 kg over 12 months), lassitude, transient thrombocytopenia, and opportunistic infections with Varicella zoster, Pneumocystis carinii, and Mycobacterium aviumintracellulare. The patient was not homosexual and had no history of intravenous drug abuse. Immunologic studies showed a persistent lymphopenia with reversal of helper/ suppressor-cytotoxic T-lymphocyte ratios, depression of human natural killer cell function, and in-vitro lymphocyte proliferatiye responses to mitogens and viral antigens. Serum IgA levels were also elevated. Serum antibodies against cytomegalovirus, herpes simplex viruses 1 and 2, Epstein-Barr virus. Varicella zoster, and hepatitis B virus were shown, suggesting previous infection by these agents. Reactivation of cytomegalovirus infection was suggested by a rising titer of antibodies against cytomegalovirus concurrent with pneumocystis pneumonia, and was confirmed by the growth of this virus in a throat culture 2 months later. T H E ACQUIRED IMMUNODEFICIENCY SYNDROME with
life-threatening opportunistic infections has recently been described in homosexual men (1-7), drug abusers (2, 4, 5, 7, 8), and Haitian refugees (4, 5, 9). The common variables leading to the acquisition of the syndrome among these groups are now under intensive scrutiny and investigation. Recently, the acquired immunodeficiency syndrome with fatal complications was recognized in three previously healthy patients with hemophilia living in Westchester County, New York; Denver, Colorado; and northeastern Ohio (5, 10). None of the patients were homosexual or had a history of intravenous drug abuse. We report a fourth patient with hemophilia from Alabama with this syndrome and life-threatening opportunistic infections. Materials and Methods IMMUNOLOGIC STUDIES
Mononuclear cells were isolated from heparinized peripheral blood on ficoU-hypaque gradients, and cell preparations were quantitated by indirect immunofluorescence using an aflinity• From the Division of Hematology/Oncology, Department of Medicine, Cellular Immunobiology Unit, Tumor Institute, Comprehensive Cancer Center, Division of Virology, Department of Pediatrics, University of Alabatna in Birtninghatn; Hematology/Oncology Service. Birmingham Veterans Administration Medical Center; and the Birmingham Hemophilia Program, Alabama State Crippled Children and Rehabilitation Service. Birtrjingham, Alabama. Annals of Internal Medicine. 1 983;98;287-290.
purified fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin reagent. T lymphocytes were quantitated using the following monoclonal antibodies: Leu-4 (total T), Leu-3 (T-helper), and Leu-2 (T-suppressor-cytotoxic) (Becton Dickinson, Mountainview, California). Monoclonal antibodies for quantitation of total B lymphocytes (HB-2) as well as human natural killer (HNK) cells (HNK-1 or Leu-7) were produced (11). The positive cells were enumerated by a fluorescence activated cell sorter (FACS IV; Becton Dickinson, Sunnyvale, California) and 10 000 cells were analyzed for each sample. Natural killer cell activity was assessed by means of a short-term 5'Cr release assay previously described (11). Three effector-totarget (erythroleukemia cell line K562) ratios (10:1, 5:1, and 2.5:1) were used. In-vitro lymphocyte proliferative response to mitogens or viral antigens was assayed in the following manner. Briefly, 1 X 105 isolated lymphocytes supplemented with 5% monocytes were added to triplicate wells of a microtiter plate. Cells were incubated with the test mitogen or viral antigen for 3 or 6 days respectively in 5% carbon dioxide at 37 °C. Eighteen hours before harvest the cultures were pulsed with tritiated thymidine. Incorporation of counts were ascertained by liquid scintillation counting and the results expressed as mean counts per minutes. VIROLOGIC STUDIES
Clinical specimens were processed for viral isolation by standard methods using monolayers of human diploid fibroblasts and African green monkey kidney cells (12). Antibodies (IgG) to Varicella zoster and Epstein-Barr nuclear antigens and viral capsid antigens were ascertained by established immunofluorescent procedures (12). Antibodies to cytomegalovirus were assessed by cytomegelisa (M.A. Bioproducts; Walkersville, Maryland). Antibodies to herpes simplex virus were ascertained by microneutralization tests (12). The detection of Pneumocystis carinii antigens was done by counterimmunoelectrophoresis (13). Case Report
A 55-year-old white man with classic hemophilia presented to the University of Alabama in Birmingham Medical Center in March 1982 with herpes zoster involving the ophthalmic branch of the left fifth cranial nerve and a secondary beta-hemolytic streptococcus infection. The patient had had insulin-dependent diabetes mellitus since 1978 and was on a home infusion program with factor VIII concentrates. Over the previous 6-month period that began after an influenza immunization the patient had a weight loss of 17 kg and recurrent symptoms of malaise, sore throat, hoarseness, anorexia, and nausea. At presentation, lymphopenia (0.45X10VL) and splenomegaly were also noted. The patient's serum liver enzyme levels were elevated. His infections responded to treatment with vidarabine and antibiotics. An extensive search for lymphoma was negative, and a liver biopsy specimen showed chronic persistent hepatitis. The patient did well until June 1982, when he was hospital©1983 American College of Physicians
287
Table 1. Immunologic Findings
August 1982 Total leukocyte count, X 10^ cells/L Total lymphocyte count, X 10^ cells/L T lymphocytes, % of total mononuciear cells Leu-4 (total T lymphocytes) Leu-3 (helper T lymphocytes) Leu-2 (suppressor-cytotoxic T lymphocytes) Leu 3/Leu 2 ratio (T-helper/suppressor-cytotoxic) B lymphocytes (HB-2), % of total mononuciear cells Human natural killer (HNK) cells HNK-1, % of total mononuciear cells Natural killer functionf Percent of HNK-1 + co-expressing Leu-4+ Serum immunoglobulin, mg/dL IgG IgA IgM Lymphocyte proliferative response to mitogens,
Patient Samples September 1982
October 1982
Controls* (Mean ± SD)
4.7 1.05
5.2 0.67
3.3 0.33
4.0-10.8 1.64-4.1
48 6 30 0.2 5
34 5 26 0.2 4
55 5 39 0.1 3
60 ± 1 39 ± 1 19 ± 3 2.1 ± 1.1
11 13 94
17 5 96
33 20 100
1799 714 . 181
1596 823 269
855-1800 75-295 82-245
4626 223 481
100 245 ± 2 6 638 55 293 ± 7031 11 308 ± 1508 7981 2226 1063
11.1 ± 0.6
12.1 ± 7.9 44.2 ± 15.6 40.8 ± 22.4
counts/min
Phytohemagglutinin Concanavalin A Pokeweed mitogen Cytomegalovirus Herpes simplex 1 Varicella zoster
28 493 18 127 1755 260 133 270
•Represents values obtained in this laboratory from 39 healthy persons between 20 and 60 years old. In addition, a normal control was always assayed with each sample to confirm the validity of the results. For lymphocytic proliferative response to viral antigens, only values for concurrently assayed normal controls are given. •fPercent of " C r release at effector-to-target (erythroleukemia cell line K562) ratio of 10:1.
ized after 3 days of nonproductive cough, temperature of 39.5 °C, and respiratory distress with hypoxemia. The patient had had an additional 20-kg weight loss. A chest roentgenogram showed diffuse bilateral lower-lobe infiltrates, and the patient needed 2 weeks of ventilatory support. Numerous cultures of blood and sputum failed to identify the cause. Cultures and special staining done on a transbronchial lung biopsy specimen did not identify any organisms. The patient improved on cephapirin sodium, gentamycin, and erythromycin, and resolution of infiltrates was seen on chest roentgenogram. He was discharged after 17 days. Cutaneous candidiasis involving the right axilla and groin developed while the patient was hospitalized and resolved with topical mycostatin treatment. After being discharged, the patient continued to have lassitude and weight loss despite nutritional support. Lymphopenia persisted and transient thrombocytopenia (63X1OVL) was noted in July 1982. In August 1982, a reversal of helper/suppressor-cytotoxic T-cell ratio (Table 1) was documented while the patient was asymptomatic. In September 1982, the patient had respiratory distress, fever, hypoxemia, and bilateral pneumonic infiltrates, lost another 10 kg of weight, and was readmitted to the hospital. Erythromycin and trimethoprim-sulfamethoxazole therapy, administered orally, was initiated on the second hospital day but the patient continued to deteriorate, and ventilatory support was again needed. An open lung biopsy specimen obtained on the fifth hospital day showed abundant P. carinii. Viral cultures of lung tissue were negative, and suspended cells from the tissue failed to stain with fluorescein conjugated monoclonal antibodies to cytomegalovirus or herpes simplex viruses 1 and 2. The patient recovered on intravenous trimethoprim-sulfamethoxazole, 320 mg/6 h, was extubated by the 11th day, and was discharged on the 14th day. Pneumocystis caririii antigen was detected by counterimmunoelectrophoresis in serum obtained 2 days before the open lung biopsy but was not found in serum obtained 28 days later. A bacterial culture of the biopsy specimen was positive 9 weeks later growing Mycobacterium avium-intracellulare. At this time the patient had no pulmonary symptoms, and a chest roentgenogram was normal. 288
March 1983 • Annals of Internal Medicine • Volume 98 * Number 3
In October 1982, the patient was hospitalized after having diarrhea for 2 weeks. No gastrointestinal condition or causative organism was found, and the diarrhea resolved spontaneously. Ascites with hepatosplenomegaly was seen while the patient was hospitalized. Bacterial and viral cultures of the transudative ascitic fluid were negative, as were findings on cytologic examination. The ascites was thought to be secondary to cirrhosis, and no further investigation was pursued. During and after these two periods of hospitalization, lymphopenia and perturbation of cell-mediated immunity with reversal of helper/suppressor-cytotoxic T-cell ratio persisted (Table 1). In-vitro lymphocyte proliferative responses to phytohemagglutinin, concanavalin A, pokeweed mitogen, cytomegalovirus, herpes simplex 1, and Varicella zoster were shown to be depressed as compared to control values. Skin testing showed no reactivity to Trichophyton, mumps, purified protein derivative, and Candida. Viral cultures of stool, urine, throat, sputum, and ascitic fluid were negative until November 1982 when a throat culture grew cytomegalovirus (Table 2). The presence of antibodies to cytomegalovirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, varicella zoster virus, and hepatitis B surface and core antigens showed previous infections. Evidence of cytomegalovirus infection dates from 1976, that of Epstein-Barr virus and varicella zoster virus infections from 1978. The rising titer of antibodies against cytomegalovirus in September 1982, 2 months before a positive culture was obtained, represents a reactivation of the infection. The human leukocyte antigen phenotype of this patient was A 1, 2; B 50, 8; C w6, w7; DR w3, w4. Discussion
All four reported patients with hemophilia and the acquired immunodeficiency syndrome developed Pneumocystis carinii pneumonitis and other opportunistic infections. None of the patients were homosexual or had a history of intravenous drug abuse. Our patient was previously healthy except for the history of diabetes mellitus.
Each of the earlier findings in this patient before the development of a pneumocystis pneumonia and Mycobacterium avium-intracellulare infection, including a dramatic weight loss, development of herpes zoster infection, and the presence of lymphopenia and transient thrombocytopenia, may not have any meaning in the acquired immunodeficiency syndrome by itself, but together they constitute an important clue. We were also able to show alterations in cell-mediated immunity in this patient at least 1 month before pneumocystis infection was documented. There was a significant and persistent reversal of the helper/suppressor-cytotoxic T-cell ratio with an absolute decrease in both populations (Table 1). The percent of natural killer cells identified by the Leu-7 antibody was normal but functional natural killer activity was significantly depressed. The natural killer cells were also shown to co-express the Leu-4 (total T) marker. Abo and Balch (14) have shown the HNK-1+ OKT-3 + (HNK-1 + Leu-4+) subpopulation of cells in normal persons also had low natural killer activity. Similar to the findings in many other patients with the acquired immunodeficiericy syndrome, this patient also had a depressed proliferative response of the lymphocytes to various mitogens (2, 3, 6, 8) and a polyclonal elevation of levels of serum IgA (2, 3, 6, 8). The cause of the acquired immunodeficiency syndrome atnong patients with hemophilia is unknown. All four patients had received large quantities of commercial factor VIII concentrates. To postulate a responsible vector in the blood products is an attractive hypothesis, but definite proof is lacking. Of viruses that may be carried in blood products, cytomegalovirus, Epstein-Barr virus and hepatitis B virus may cause transient alteration of lymphocyte functions and reversal of the helper/suppressorcytotoxic T-cell ratio (15-20), but none have been unequivocally linked to the development of this syndrome. Whether a repeat exposure to these agents may be responsible for the persistent reversal of the helper/suppressor-cytotoxic T-cell ratio in this patient is unknown. Our patient had positive titers of serum antibodies to various viruses (Table 2). Serum antibodies against cytomegalovirus have been detected since 1976, and the rising titers concurrent with Pneumocystis carinii pneumonia represent a reactiviation of the infection, and was confirmed 2 months later when a throat culture grew cytomegalovirus. Although single donor and commercial pooled blood products have been available to patients with hemophilia for over 10 years, the acquisition of the acquired immunodeficiericy syndrome by these patients has only been recognized recently. We are uncertain if there is a link between patients with hemophilia and other populations at risk, including homosexual men, drug abusers, and Haitian refugees. If a vector is responsible, how this is transmitted among these populations and, in particular, whether the transmission to patients with hemophilia is from the | other populations at risk and through blood products become extremely important issues. I
ACKNOWLEDGMENTS: The authors thank Dr. Max D. Cooper for support and advice during the study and critical review of the manuscript. Dr.
Table 2. Virologic Findings
Serologic tests Cytomegalovirus
Dates
Results
20 October 1976 11 May 1978 15 September 1982 13 October 1982
1:16 1:16 1:256 1:256
Epstein-Barr virus Viral capsid antigen 11 May 1978 29 July 1982 15 September 1982 Nuclear antigen 29 July 1982 15 September 1982 Herpes simplex viruses 1 and 2 15 September 1982 Varicella zoster virus 11 May 1978 15 September 1982 Hepatitis B virus Surface antigen 30 April 1975 2 December 1976 26 March 1982 Surface antibody 1 April 1982 15 September 1982 Core antibody 15 September 1982 Culture specimens Stool 15 September 1982 Urine Sputum Throat Lung biopsy 17 September 1982 Urine 13 October 1982 Throat 22 October 1982 Stool Ascites fluid Throat 15 November 1982
1:64 1:64 1:64 1:16 1:16 1:1024 1:64 1:64 Negative Negative Negative Positive Positive Positive Negative Negative Negative Negative Negative* Negative Negative Negative Negative Positive for cytomegalovirus
•Staining with monoclonal antibodies against cytomegalovirus. and herpes simplex 1 and 2 viruses were also negative.
William Gathings for supplying the immunoglobulin reagent, Dr. Toru Abo for doing the human natural killer cell studies. Dr. Linda Pifer (University of Tennessee, Memphis, Tennessee) for doing the Pneumocystis carinii anugen detection, and Mrs. Sharon Garrison for assistance in preparation of the manuscript. Grant support: in part by grants CA 16673 from the National Cancer Institute: 5MO1-RR32 from the National Institutes of Health; HD-106999 from the National Institute of Child Health and Human Development; and Veterans Administration Project MRIS 7133. • Requests for reprints should be addressed to Man-Chiu Poon, M.D.; Divisin of Hematology/Oncology. Department of Medicine, tjniversity of Alabama in Birmingham; Birmingham, AL 35294. References 1. GoTTLtEB MS, ScHROFF R, ScHANKER HM, et al. Pneumocystitis carin//pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency. N Engl J Med. 1981;305:1425-31. 2. MASUR H , MiCHELts MA, GREENE JB, et al. An outbreak of communi-
ty-acquired Pneumocystis carinii ptieumonia: initial manifestatioti of cellular immune dysfunction. N Engl J Med. 1981;305:1431-8. 3. StEGAi. FP, LOPEZ C, HAMMER G S et al. Severe acquired immunodeficiency in male homosexuals, manifested by chronic perianal ulcerative Herpes simplex lesions. N Engl J Med. 1981;305:1439-44. 4. AuERBACH DM, BENNETT JV, BRACHMAN PS, et al. Report of the
Center for Disease Control Task Force on Kaposi's Sarcoma and Opportunistic Infections: epidemiologic aspects of the current outbreak of Kaposi's sarcoma and opportunistic infections. A^ Engl J Med. 1982;306:248-52. 5. CENTERS FOR DISEASE CONTROL. Update on acquired immune defi-
ciency syndrome (AIDS)—United States. Morbid Mortal Weekly Rep. '1982;31:507-8, 513-4. 6. STAHL RE,. FRIEDMAN-KIEN A, DUBtN R, MARMOR M , ZOLLA-PAZ-
NER S. Immunologic abnormalities in homosexual men: relationship to Poon et al. • Acquired Immunodeficiency Syndrome
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Kaposi's sarcoma. Am J Med. 1982;73:171-8. 7. GREENE JB, SIDHU GS, LEWIN S, et al. Mycobacterium avium-intracel-
lulare: a cause of disseminated life-threatening infection in homosexuals and drug abusers. Ann Intem Med. 1982;97:539-46. 8. MASUR H , MICHELIS M A , WORMSER G P , et al. Opportunistic infection
in previously healthy women: initial manifestations of a community-acquired cellular immunodeficiency. Ann Intern Med. 1982;97:533-9. 9. HENSLEY G T , MOSKOWITZ LB, PITCHENIK A E , et al. Opportunistic
infections and Kaposi's sarcoma among Haitians in the United States. Morbid Mortal Weekly Rep. 1982;31:353-4, 360-1. 10. EHRENKRANZ NJ, RuBiNi J, GONN R, et al. Pneumocystis carinii pneumonia among persons with hemophilia A. Morbid Mortal Weekly Rep. 1982;31:365-7. 11. ABO T, COOPER MD, BALCH CM. Characterization of HNK-1+ (Leu-
7) human lymphocytes; I. Two distinct phenotypes of human NK cells with different cytotoxic capability. J Immunol. 1982;129:1752-7. 12. REYNOLDS D W , STAGNO S, ALFORD CA. Laboratory diagnosis of cytomegalovirus infections. In: LENETTE EH, SCHMIDT NJ, eds. Diagnostic
Procedures for Viral Rickettsial and Chlamydial Infection. 5th ed. Washington, D.C: American Public Health Association, Inc.; 1979:399439. 13. PIFER L L , HUGHES W T , STAGNO S, WOODS D . Pneumocystis carinii
infection: evidence for high prevalence in normal and immuno-sup-
pressed children. Pediatrics. 1978;61:35-41. 14. ABO T, BALCH CM. Characterization of HNK-1+ (Leu-7) human lymphocytes: II. Distinguishing phenotypic and functional properties of natural killer cells from activated NK-like cells. / Immunol. 1982; 129:1758-61. 15. NoTKiNS AL, MERGENHAGEN SE, HOWARD RJ. Effect of virus infections on the function of the immune system. Annu Rev Microbiol. 1970;24:525-38. 16. REINHERZ E L , O'BRIEN C, ROSENTHAL P , SCHLOSSMAN S F . The cel-
lular basis for viral-induced immunodeficiency: analysis by monoclonal antibodies. J Immunol. 1980;125:l269-74. 17. RiNALDO CR JR, CARNEY WP, RICHTER BS, BLACK P H , HIRSCH MS.
Mechanisms of immunosuppression in cytomegaloviral mononucleosis. J Infect Dis. 198O;141:488-95. 18. CARNEY WP, RUBIN R H , HOFFMAN R A , HANSEN WP, HEALEY K ,
HIRSCH MS. Analysis of T lymphocyte subsets in cytomegalovirus mononucleosis. J Immunol. 1981; 126:2114-6. 19. RUBIN RH, CARNEY WP, SCHOOLEY R T , et al. The effect of infection
of T-lymphocyte subpopulations: a preliminary report. Int J Immunopharmacol. 1981;3:3O7-12. 20. THOMAS HC. T-cell subsets in patients with acute and chronic HBV infection, primary biliary cirrhosis and alcohol induced liver disease. Int J Immunopharmacol. 1981;3:301-5.
The Acquired Immunodeficiency Syndrome and Mycobacterium aviumintracellulare Bacteremia in a Patient with Hemophilia JAMES L. ELLIOTT, M.D.; WILLIAM L HOPPES, M.D.; MARVIN S. PLATT, M.D.; JOHN G. THOMAS, Ph.D.; INDRAVADAN P. PATEL, M.D.; and ALI GANSAR, M.D.: Canton and Akron, Ohio A 27-year-old previously healthy man with hemophilia presented with Pneumocystis car/n/V pneumonia. The patient had several episodes of oral candidiasis followed by disseminated infection with Mycobacterium aviumintracellulare. He was not homosexual nor did he take illicit drugs, but he had been self-administering two to four monthly infusions of factor VIII concentrate for 7 years, invitro lymphocyte studies showed findings consistent with the acquired immunodeficiency syndrome that had previously been reported only in homosexual men, drug addicts, and Haitian refugees. The cause of this syndrome is unknown, but the possibility that it is associated with a transmissible agent acquired through the use of blood products such as factor VIII concentrate must be considered. IN THE PAST YEAR there have been reports of severe opportunistic infections, persistent generalized lymphadenopathy, malignancies, and autoimmune diseases in previously healthy homosexual men, drug addicts, and Haitian refugees (1-8). Many of these patients have a profound defect in cellular immune function with retention of normal humoral immune response. The cause of this acquired immunodeficiency syndrome is not known. Possible causes include immunosuppression from cytomegalovirus, Epstein-Barr virus, or another unidentified viral agent; and immunosuppression related to drug abuse, or some other transmissible factor such as a toxin. The possibility that this syndrome may be transmitted by • From the Departments of Internal Medicine and Pathology, Northeastern Ohio Universities College of Medicine, Affiliated Hospitals, Canton, Ohio; and the Department or Pathology. Children's Hospital Medical Center, Akron. Ohio. 290
Annals of Internal Medicine. 1 983;98:290-293.
commercially available blood products is suggested by reports to the Centers for Disease Control of three patients with hemophilia, none of whom were homosexual or used illicit drugs, who have developed a similar syndrome (9). A recent Centers for Disease Control report has raised the number of patients with hemophilia with the syndrome to a total of eight (10). We describe one of the three original cases. Case Report A 27-year-old white man with hemophilia A had been selfadministering two to four monthly infusions of factor VIII concentrate for the past 7 years. The patient had no history of exposure to other blood products, illicit drug use, or homosexual contacts. The patient had been well until July 1981 when he had insidious onset of dysuria progressing to fever, chills, night sweats, weakness, increasing dyspnea, cough productive of white sputum, anorexia, and a 7-kg weight loss. In August 1981 a chest roentgenogram showed pulmonary infiltrates, and the patient was treated with oral ampicillin. When he was seen in the Aultman Hospital Emergency Department in September 1981 he appeared cachectic and notably dyspneic with a temperature of 37.2° C. There were decreased breath sounds and fine rales throughout both lung fields. An initial chest roentgenogram showed bilateral pulmonary infiltrates. The patient's hemoglobin was 10.7 g/dL, and the leukocyte count was 4200/ mm3 with 50% polymorphonuclear leukocytes, 2% band forms, 36% lymphocytes, and 12% monocytes. Blood, urine, and sputum cultures were normal. Indirect fluorescent IgG titers against cytomegalovirus and Toxoplastna were positive at dilutions of 1:64 and 1:16 respectively. The IgM titers for both were negative. The IgG Epstein-Barr virus titers were 1:8192; IgM titer was negative. Serum radioimmunoassay was positive for hepatitis B surface antibody and core antibody, and negative © 1983 American College of Physicians
