Therapy for Disseminated Coccidioidomycosis
with
Transfer Factor from a Related Donor
RUSSELL W. STEELE, M.D. BARRY E. SIEGER, M.D. THEODORE R. McNITT, M.D. LAYNE 0. GENTRY, M.D. WILLIAM L. MOORE, Jr., M.D. Fort Sam Houston, Texas
From the Section of Infectious Diseases, Departments of Medicine and Pediatrics, Brooke Army Medical Center, Fort Sam Houston, Texas. This work was supported in part by research grant No. C-42-74 from the Clinical Investigation Unit of Brooke Army Medical Center. The opinions or assertions contained herein are the private ones of the authors and are not to be interpreted as official or reflecting the view of the Department of the Army. Requests for reprints should be addressed to Dr. Russell W. Steele. Infectious Diseases and Immunology, Department of Pediatrics, Brooke Army Medical Center, Fort Sam Houston, Texas 78234. Manuscript accepted October 22. 1975.
A 17 year old Caucasian woman in whom disseminated coccidioidomycosis developed with culture positive meningitis during her third trimester of pregnancy was treated with amphotericin B and subsequently with transfer factor prepared from her father’s peripheral lymphocytes. Clinical response and in vivo and in vitro Immunologic data indicated that this transfer factor afforded a significant contribution to her survival whereas previous therapy with transfer factor from an unrelated donor provided only transient immunologic+‘eactivity. This experience suggests that transfer factor prepared from a related donor with positive responses to C. immitis may be more efficacious than that prepared from an unrelated donor. Coccidioidomycosis, if contracted during pregnancy, is associated with a much higher risk of dissemination and a more severe prognosis than the disease in a nonpregnant patient [l-3]. Moreover: both the incidence of dissemination and mortality increase with increasing gestation [ 1,2]. Prior to the use of amphotericin B, mortality approached 100 per cent when disseminated coccidioidomycosis developed during the third trimester of pregnancy: and even with the introduction of amphotericin B mortality has remained high [l-4]. In the patient we describe disseminated coccidioidomycosis developed with culture positive meningitis during her last trimester of pregnancy, and therefore she presented with a minimal chance of survival. The patient was treated with amphotericin B, transfer factor from an unrelated donor, and later transfer factor prepared from her father. Sustained remission has been achieved with 15 months post-treatment follow-up now available. It was believed both by clinical response and by in vivo and in vitro laboratory data that the transfer factor prepared from her father afforded a significant contribution to her survival. MATERIALS AND METHODS LymphocyteBlastogenesis. In vitro determination coccidioidin,
as well as other control antigens,
of cellular immunity to was accomplished by mea-
suring lymphocyte blastogenesis following incubation with the antigens under
investigation. Coccidioidin was obtained as a commercial lot from Cutter Laboratories and this preparation was dialyzed six times against 200 volumes
August 1976
The American Journal of Medicine
Volume 61
263
THERAPY FOR DISSEMINATED
COCCIDIOIDOMYCOSIS-STEELE
ET AL.
of 0.15 M sodium chloride for 48 hours to remove preservatives. The material was stored at -20°C until used. Maximum in vitro stimulation of lymphocytes was obtained with a 16000 dilution: sensitivity of this material was examined in a previous communication [5]. Lymphocytes were separated from 5 ml of peripheral whole blood by a modification of the Hypaque-Ficoll gradient technic. The cell suspension was washed twice in culture medium and the lymphocytes were adjusted to a concentration of 2 X IO6 ml in RPMI 1840 medium with Hepes buffer containing 100 units of penicillin/ml, 100 pg of streptomycin/ml and 20 per cent autologous plasma. Using a biopipet, 0.1 ml of the lymphocyte suspension (2 X lo5 lymphocytes) was mixed with an equal volume of antigenic material in one well of sterile flat-bottomed microtest plates. All cultures were prepared in triplicate along with control cultures containing lymphocytes incubated with medium alone. The plates were covered with sterile plastic lids, and cultures were incubated for five days. Twenty-four hours prior to harvest, 0.05 ml of RPM 1640 medium containing 1 &i of tritiated(3HFthymidinewas added to each well. A harvesting apparatus previously described [6] was employed for the separation of reacting cells on glass fiber filters, for washing of these cells and for recovery of the radioactive material incorporated by stimulated lymphocytes. The glass fiber discs were then dried in an oven, and transferred to vials containing 5 ml of scintillation fluid for counting in a Beckman liquid scintillation spectrometer. The average counts/minute (cpm) of triplicate samples were dstermined and results were expressed as a blastogenic index: cpm of 3H-thymidine uptake for lymphocytes incubated with antigen divided by uptake following incubation with medium alone: blastogenic index =
cpm for lymphocytes + coccidioidin cpm for lymphocytes •l- medium
Based on previous studies, a blastogenic index 1 3.0 was considered a positive response. Transfer Factor. Dialyzable transfer factor was prepared and purified by the methods of Lawrence and Al-Askari [7]. Approximately 500 cc of whole blood were obtained by venipuncture, and lymphocytes were separated from the cell pack using a Hypaque-Ficoll gradient, centrifuging at 4OC to maintain optimal recovery of transfer factor. The lymphocytes were freeze-thawed in the presence of DNase 10 times, and subsequently dialyzed and concentrated by lyophilization. Potency was confirmed by passive transfer of 0.1 ml intradermally into negative human recipients followed in 72 hours by skin testing in the same site as the transfer factor injection. By this method, multiple antigens were examined with only those which showed positive skin reactivity in the donor being positive in the recipients. Induration in a previously negative recipient was greater than 5 mm to a I:10 dilution of COCcidioidin for both transfer factor preparations.
CASE REPORT Our patient was a 17 year old caucasian housewife, living in North Carolina. One week prior to delivery of her first child, she experienced fever, malaise and shortness of breath which spontaneously remitted. She had an uncomplicated
284
August 1976
labor and delivered a normal infant. On the first postpartum day, fever developed with temperature to 103.8’F. The clinical impression was endometritis, and sodium ampicillin therapy was instituted followed by a reported defervescence of fever. One week later, she again became febrile and at this time was noted to have bilateral pulmonary parenchymal nodular infiltrates and splenomegaly. She was treated with sodium cephalothin without response. Because of persistence of fever and headache, a lumbar puncture was performed. The cerebrospinal fluid contained 49 white blood cells, 39 neutrophils and 100 lymphocytes; protein and glucose were normal. On historic review, it was found that during her second trimester she had traveled by car to California, during which time she had spent 2 hours in the San Joaquin Valley area. During the third trimester of pregnancy, she had received a package of clothing with several articles wrapped in dusty newspaper sent from relatives in the San Joaquin Valley. Either incident might have represented fomite transmission of C. immitis. A repeat lumbar puncture two weeks postpartum revealed 833 white blood cells, 71 per cent neutrophils, 29 per cent lymphocytes, glucose of 24 mg/lOO ml, and protein 89 mg/lOO ml. Complement fixation titers to C. immitis were positive in this cerebrospinal fluid at titers of I:20 with a concomitant serum titer of 1:320. C. immitis was also cultured from this sample of spinal fluid. The patient was transferred to Brooke Army Medical Center for further evaluation and treatment. On arrival, examination documented the findings of intermittent disorientation, papilledema, pupillary asymmetry, right-sided central facial weakness, right ninth cranial nerve palsy, weakness of the lower right extremity and clonus of the lower left extremity. A four vessel cerebral arteriogram revealed dilatation of the lateral ventricles. Skin tests to monilia, mumps and tetanus toxoid demonstrated greater than 10 mm indurationbut a skin test for C. immitis (coccidioidin U.S.P., 1: 10, Cutter Laboratories, Berkely, Calif.) was negative. In vitro lymphocyte blastogenic responses to the mitogens, phytohemagglutinin, pokeweed, concanavalin A were positive as were the responses to the antigens, monilia, mumps and tetanus toxoid. In vitro lymphocyte blastogenic responses to coccidioidin were negative. The patient was started on amphotericin B intravenously, increasing the dose to 30 mg three times per week. Large doses were associated with marked idiosyncratic reactions, especially hypotension. Intravenous therapy was continued for 10 months at which time she had received a total dose of 4,500 mg. The administration of amphotericin B was also started intracisternally, and the dose was gradually increased to 0.3 mg twice weekly. She received a total of 6.7 mg over four months by this route. Attempts to use an Ommya reservoir for intraventricular therapy failed because of infection of one reservoir with Staphylococcus epidermidis and malfunction of a second apparatus. Despite the schedule of amphotericin B therapy, the patient’s condition progressively deteriorated. Although her cerebrospinal fluid cultures of C. immitis became negative following institutionof amphotericin B therapy, her complement fixation titers in serum stabilized at 1:128 and in the cerebrospinal fluid at 1:8. Because of this poor clinical response and the persistent antibody titers, it was elected to treat the patient with human dialyzable transfer factor. The donor of the transfer factor had had pulmonary
The American Journal of Medicine Volume 61
THERAPY FOR DISSEMINATED COCCIDIOIDOMYCOSIS-STEELE
TABLE
I
ET AL.
In Vivo and In Vitro Evaluation of Cellular Immunity During Two Courses of Transfer Factor Therapy -___.__ 1973 1974 ______.. _.. .__
--.___-..
Nov. 2Dec. 15
Dec. 15
Dec. 22
-
+
+
Dec. 29 Jan. 5
Jan. 8Feb. 1
Feb. 1
Feb. 7
Feb. 14
Mar.
Apr.
MayAug.
Sept.
_
+
+
+
_
_
_
_
..--
Therapy Transfer factor Amphoterrcin % intravenous intracisternal Skin tests* Cocci Monilia Blastogenesrs+ Cocci Monilia Antibody Serum Cerebrosprnal
Unrelated
+
+
donor
Related donor ._.~_____
4
___-.
.-... _------~-._---)
.W
* 0 13 0.6-I .1 3.8-10.7 1:3X-1:160 I:20
fluid Cerebrospinal
+
7 17
4 ND
0 11
0 ND
13.7 8.1
5.3 8.5
2.6 11.8
1.9 7.4
0 12 0.9-I .5 6.7-9.9
3 15
8 ND
17 12
>15 .15
315 >I5
>I5 >15
>15 >15 10.9 17.2
8.9 12.0
11.2 17.7
5.5 10.8
5.7-l 1.4 9.4-16.8
13.3 15.2
9.0-l 5.1 8.5-14.7
1:128 1:8
I:128 1:8
ND ND
1:128 I:8
ND ND
1:128 1:8
ND ND
ND ND
I:32 0
1:16 0
1 :16 0
-
_
ND
-
ND
_
ND
ND
_
-
_
I:8 0 -
culture NOTE: Rules indicate duration of therapy. ND = not done. Skin test reactions to coccidioidin 1 :lO for the donors were: unrelated donor 37 mm induration, related donor 43 mm and to monilia 1 :I00 were: unrelated donor 21 mm, related donor 17 mm. *Skin tests were applied 72 hours after the dose of transfer factor was given and were measured as millimeters of induration at 48 hours. + Expressed as a blastoqenic index: cpm of ‘H-thymidine uptake for lymphocytes incubated with antigen divided by uptake following incubation
with medium
alone.
coccidioidomycosis at age 13. He had remained skin test positive and when intradermally tested with soluble coccidioidin reacted with 8 mm of indurationusing a 1:1000 dilution and 37 mm of indurationwith a 1:lO dilution. These skin tests were recorded at 48 hours. The patient was given 1 unit (equivalent to 1 X log lymphocytes) of transfer factor subcutaneously at weekly intervals for four consecutive weeks (Table I). Three days after receiving the first unit she was retested, at which time the skin test response to 1: IO coccidioidin demonstrated induration of 6 by 7 mm. In vitro lymphocyte blastogenic responses increased from a blastogenic index of 1.07 to 13.7 at this time. These responses, however, were transient, and both in vivo and in vitro measurements reverted to negative despite the administration of additional transfer #actor. The patient deteriorated neurologically as a result of obstructive hydrocephalus and a ventriculoperitoneal shunt was placed. Three months after her admission, it was learned that her father had had pulmonary coccidioidomycosis at age 13. He was strongly positive to a 1:iO dilution of coccidioidin intradermally reacting with 43 mm of induration. Dialyzable transfer factor was prepared from the patient’s father as previously described and the patient was given 1 unit subcutaneously each week for three consecutive weeks. The coccidioidin skin tests after the first unit were positive at 8 mm of induration and following the third unit increased to 17 mm of induration. Her blastogenic index after administration of the first unit increased from 1.45 to 8.9. Both the skin test and the blastogenic index remained positive over the eight months they were followed. Following
therapy with transfer
factor from her father, the patient experienced gradual but progressive improvement over four months. Her condition then stabilized
and no further
significant
improvement
curred over an additional 15 months of observation.
oc-
COMMENTS The more severe prognosis for coccidioidomycosis in the pregnant patient is most likely attributable to the known transient depression of cell mediated immunity demonstrated during gestation [8]; the risk of dissemination then increases to an estimated 20 per cent, 100 times that of the nonpregnant white woman and three times that of the nonpregnant black [2]. A majority of reported cases have occurred during the third trimester [l-3]. The exact mechanism of this maternal response remains poorly understood but is apparently directed to protect the developing fetus from rejection. Such tolerance during pregnancy may carry over to newly introduced foreign antigens such as fungal antigens with resultant proliferation of suppressor T cells and subsequent depression of cell mediated immune reactions to the invading organisms [9]. Accepting this hypothesis, the role of immunotherapeutic products like transfer factor can better be explained. Another suggested mechanism is the elaboration of an inhibitory factor into the serum during gestation which depresses cellular immunity [lo]; in the present studies, no such factor could be demonstrated since C. immitis blastogenie assays for known positive controls were not decreased in the presence of the patient’s serum, nor were her responses any different when comparing AB positive serum or fetal calf serum to autologous serum as the medium supplement. The present case illustrates the associations between pregnancy, disseminated coccidioidomycosis and defects in cellular immunity. In this patient the defect
August 1976
The American Journal of Medicine Volume 61
265
THERAPY FOR DISSEMINATED COCCIDIOIDOMYCOSIS-STEELE
ET AL
appeared specific to C. immitis since in vivo and in vitro reactivity to other fungal, viral and bacterial antigens as well as nonspecific in vitro stimulation with mitogens was normal. A therapeutic effect was achieved when coccipositive transfer factor from a related donor was combined with amphotericin B. No apparent clinical response and only transient conversbn of cellular immune responses was seen with amphoteiicin B and coccipositive transfer factor from the un&lated donor during a four month period of observation:” It is of course not certain that the clinical benefit was related primarily to the second series of transfer factor but most evidence, as observed,‘suggests this. This question then remains whether the fact that the transfer factor donor was related to the recipient was a significant factor in success of therapy. The skin test and in vitro responses of the two transfer factor donors were comparable and therefore indicate that potency of transfer factor as judged by donor responses alone could not account for the differences in efficacy. A number of clinical reports have attested to the efficacy of transfer factor for severe coccidioidomycosis
as well as for other infectious processes such as chronic mucocutaneous candidiasis, and available information for transfer factor therapy has been summarized previously [ 111. A recent review from the Coccidioidomycosis Cooperative Treatment Group (CCTG) represents the largest series of patients, 42, treated with transfer factor for coccidioidomycosis [ 121 and the present case was it$uded in this study. Clinical and immunologic responses were variable for the 42 patients, but data did suggest clinical efficacy of transfer factor. More definitive information hopefully will be obtained by the CCTG in a controlled prospective trial of placebo transfer factor versus coccidioidomycosis positive transfer factor currently in progress. Finally, in studies employing transfer factor as immunotherapy for patients with osteogenic sarcoma, donors with similar environmental exposure appear preferable, and these are often relatives [ 131. The present report suggests that both the environmental exposure to the agent, in this case C. immitis, as well as the donor being related to the recipient of transfer factor may be of importance for efficacy of transfer factor therapy.
REFERENCES 1.
2.
3.
4. 5.
6.
7.
286
Smale LE, Waechter KG: Dissemination of coccidioidomycosis in pregnancy. Am J Obstet Gynecol 107: 356, 1970. Harris RE: Coccidioidomycosis complicating pregnancy. Report of 3 cases and review of the literature. Obstet Gynecol28: 401, 1966. Hadsall FJ, Acquarelli MJ: Disseminated coccidioidomycosis presenting as facial granulomas in pregnancy: a report of two cases and a review of the literature. The Laryngoscope 83: 51, 1973. Pappagianis D: Personal communication, 1975. Steele RW, Cannady PB Jr, Moore WC Jr, Gentry LO: Skin test and blastogenic responses to sporothrix schenckii. J Clin Invest 57: 156, 1976. Steele RW, Hensen SA, Vincent MM, Fuccillo DA, Bellanti JA: A 51Cr microassay technique for cell-mediated immunity to viruses. J lmmunol 110: 1502, 1973. Lawrence HS: Transfer factor. Adv lmmunol 11: 195. 1969.
August 1976
The American Journal of Medicine
8.
9. 10.
11.
12. 13.
Volume 61
Thong YH, Steele RW, Vincent MM, Hensen SA. Bellanti JA: Impaired in-vitro cell-mediated immunity to rubella virus during pregnancy. N Engl J Med 289: 604, 1973. Kantor FS: Infection, anergy and cell-mediated immunity. N Engl J Med 292: 629, 1975. Ceppellini R, Bonnard GD, Coppo F: Mixed leukocyte cultures and HL-A antigens. I. Reactivity of young fetuses, newborns and mothers at delivery. Transplant Proc 3: 58, 197 1. Kirkpatrick CH, Gallin JI: Treatment of infectious and neoplastic diseases with transfer factor. Oncology 29: 46, 1974. Graybill JR: Transfer factor in coccidioidomycosis. Clin Res 23: 304. 1975. Levin AS, Byers VS, Fudenberg HH, Wybran J, Hackett AJ, Johnston JO, Spitler LE: Osteogenic sarcoma: immunologic parameters before and during immunotherapy with tumor-specific transfer factor. J Clin Invest 55: 487, 1975.
with
Transfer Factor from a Related Donor
RUSSELL W. STEELE, M.D. BARRY E. SIEGER, M.D. THEODORE R. McNITT, M.D. LAYNE 0. GENTRY, M.D. WILLIAM L. MOORE, Jr., M.D. Fort Sam Houston, Texas
From the Section of Infectious Diseases, Departments of Medicine and Pediatrics, Brooke Army Medical Center, Fort Sam Houston, Texas. This work was supported in part by research grant No. C-42-74 from the Clinical Investigation Unit of Brooke Army Medical Center. The opinions or assertions contained herein are the private ones of the authors and are not to be interpreted as official or reflecting the view of the Department of the Army. Requests for reprints should be addressed to Dr. Russell W. Steele. Infectious Diseases and Immunology, Department of Pediatrics, Brooke Army Medical Center, Fort Sam Houston, Texas 78234. Manuscript accepted October 22. 1975.
A 17 year old Caucasian woman in whom disseminated coccidioidomycosis developed with culture positive meningitis during her third trimester of pregnancy was treated with amphotericin B and subsequently with transfer factor prepared from her father’s peripheral lymphocytes. Clinical response and in vivo and in vitro Immunologic data indicated that this transfer factor afforded a significant contribution to her survival whereas previous therapy with transfer factor from an unrelated donor provided only transient immunologic+‘eactivity. This experience suggests that transfer factor prepared from a related donor with positive responses to C. immitis may be more efficacious than that prepared from an unrelated donor. Coccidioidomycosis, if contracted during pregnancy, is associated with a much higher risk of dissemination and a more severe prognosis than the disease in a nonpregnant patient [l-3]. Moreover: both the incidence of dissemination and mortality increase with increasing gestation [ 1,2]. Prior to the use of amphotericin B, mortality approached 100 per cent when disseminated coccidioidomycosis developed during the third trimester of pregnancy: and even with the introduction of amphotericin B mortality has remained high [l-4]. In the patient we describe disseminated coccidioidomycosis developed with culture positive meningitis during her last trimester of pregnancy, and therefore she presented with a minimal chance of survival. The patient was treated with amphotericin B, transfer factor from an unrelated donor, and later transfer factor prepared from her father. Sustained remission has been achieved with 15 months post-treatment follow-up now available. It was believed both by clinical response and by in vivo and in vitro laboratory data that the transfer factor prepared from her father afforded a significant contribution to her survival. MATERIALS AND METHODS LymphocyteBlastogenesis. In vitro determination coccidioidin,
as well as other control antigens,
of cellular immunity to was accomplished by mea-
suring lymphocyte blastogenesis following incubation with the antigens under
investigation. Coccidioidin was obtained as a commercial lot from Cutter Laboratories and this preparation was dialyzed six times against 200 volumes
August 1976
The American Journal of Medicine
Volume 61
263
THERAPY FOR DISSEMINATED
COCCIDIOIDOMYCOSIS-STEELE
ET AL.
of 0.15 M sodium chloride for 48 hours to remove preservatives. The material was stored at -20°C until used. Maximum in vitro stimulation of lymphocytes was obtained with a 16000 dilution: sensitivity of this material was examined in a previous communication [5]. Lymphocytes were separated from 5 ml of peripheral whole blood by a modification of the Hypaque-Ficoll gradient technic. The cell suspension was washed twice in culture medium and the lymphocytes were adjusted to a concentration of 2 X IO6 ml in RPMI 1840 medium with Hepes buffer containing 100 units of penicillin/ml, 100 pg of streptomycin/ml and 20 per cent autologous plasma. Using a biopipet, 0.1 ml of the lymphocyte suspension (2 X lo5 lymphocytes) was mixed with an equal volume of antigenic material in one well of sterile flat-bottomed microtest plates. All cultures were prepared in triplicate along with control cultures containing lymphocytes incubated with medium alone. The plates were covered with sterile plastic lids, and cultures were incubated for five days. Twenty-four hours prior to harvest, 0.05 ml of RPM 1640 medium containing 1 &i of tritiated(3HFthymidinewas added to each well. A harvesting apparatus previously described [6] was employed for the separation of reacting cells on glass fiber filters, for washing of these cells and for recovery of the radioactive material incorporated by stimulated lymphocytes. The glass fiber discs were then dried in an oven, and transferred to vials containing 5 ml of scintillation fluid for counting in a Beckman liquid scintillation spectrometer. The average counts/minute (cpm) of triplicate samples were dstermined and results were expressed as a blastogenic index: cpm of 3H-thymidine uptake for lymphocytes incubated with antigen divided by uptake following incubation with medium alone: blastogenic index =
cpm for lymphocytes + coccidioidin cpm for lymphocytes •l- medium
Based on previous studies, a blastogenic index 1 3.0 was considered a positive response. Transfer Factor. Dialyzable transfer factor was prepared and purified by the methods of Lawrence and Al-Askari [7]. Approximately 500 cc of whole blood were obtained by venipuncture, and lymphocytes were separated from the cell pack using a Hypaque-Ficoll gradient, centrifuging at 4OC to maintain optimal recovery of transfer factor. The lymphocytes were freeze-thawed in the presence of DNase 10 times, and subsequently dialyzed and concentrated by lyophilization. Potency was confirmed by passive transfer of 0.1 ml intradermally into negative human recipients followed in 72 hours by skin testing in the same site as the transfer factor injection. By this method, multiple antigens were examined with only those which showed positive skin reactivity in the donor being positive in the recipients. Induration in a previously negative recipient was greater than 5 mm to a I:10 dilution of COCcidioidin for both transfer factor preparations.
CASE REPORT Our patient was a 17 year old caucasian housewife, living in North Carolina. One week prior to delivery of her first child, she experienced fever, malaise and shortness of breath which spontaneously remitted. She had an uncomplicated
284
August 1976
labor and delivered a normal infant. On the first postpartum day, fever developed with temperature to 103.8’F. The clinical impression was endometritis, and sodium ampicillin therapy was instituted followed by a reported defervescence of fever. One week later, she again became febrile and at this time was noted to have bilateral pulmonary parenchymal nodular infiltrates and splenomegaly. She was treated with sodium cephalothin without response. Because of persistence of fever and headache, a lumbar puncture was performed. The cerebrospinal fluid contained 49 white blood cells, 39 neutrophils and 100 lymphocytes; protein and glucose were normal. On historic review, it was found that during her second trimester she had traveled by car to California, during which time she had spent 2 hours in the San Joaquin Valley area. During the third trimester of pregnancy, she had received a package of clothing with several articles wrapped in dusty newspaper sent from relatives in the San Joaquin Valley. Either incident might have represented fomite transmission of C. immitis. A repeat lumbar puncture two weeks postpartum revealed 833 white blood cells, 71 per cent neutrophils, 29 per cent lymphocytes, glucose of 24 mg/lOO ml, and protein 89 mg/lOO ml. Complement fixation titers to C. immitis were positive in this cerebrospinal fluid at titers of I:20 with a concomitant serum titer of 1:320. C. immitis was also cultured from this sample of spinal fluid. The patient was transferred to Brooke Army Medical Center for further evaluation and treatment. On arrival, examination documented the findings of intermittent disorientation, papilledema, pupillary asymmetry, right-sided central facial weakness, right ninth cranial nerve palsy, weakness of the lower right extremity and clonus of the lower left extremity. A four vessel cerebral arteriogram revealed dilatation of the lateral ventricles. Skin tests to monilia, mumps and tetanus toxoid demonstrated greater than 10 mm indurationbut a skin test for C. immitis (coccidioidin U.S.P., 1: 10, Cutter Laboratories, Berkely, Calif.) was negative. In vitro lymphocyte blastogenic responses to the mitogens, phytohemagglutinin, pokeweed, concanavalin A were positive as were the responses to the antigens, monilia, mumps and tetanus toxoid. In vitro lymphocyte blastogenic responses to coccidioidin were negative. The patient was started on amphotericin B intravenously, increasing the dose to 30 mg three times per week. Large doses were associated with marked idiosyncratic reactions, especially hypotension. Intravenous therapy was continued for 10 months at which time she had received a total dose of 4,500 mg. The administration of amphotericin B was also started intracisternally, and the dose was gradually increased to 0.3 mg twice weekly. She received a total of 6.7 mg over four months by this route. Attempts to use an Ommya reservoir for intraventricular therapy failed because of infection of one reservoir with Staphylococcus epidermidis and malfunction of a second apparatus. Despite the schedule of amphotericin B therapy, the patient’s condition progressively deteriorated. Although her cerebrospinal fluid cultures of C. immitis became negative following institutionof amphotericin B therapy, her complement fixation titers in serum stabilized at 1:128 and in the cerebrospinal fluid at 1:8. Because of this poor clinical response and the persistent antibody titers, it was elected to treat the patient with human dialyzable transfer factor. The donor of the transfer factor had had pulmonary
The American Journal of Medicine Volume 61
THERAPY FOR DISSEMINATED COCCIDIOIDOMYCOSIS-STEELE
TABLE
I
ET AL.
In Vivo and In Vitro Evaluation of Cellular Immunity During Two Courses of Transfer Factor Therapy -___.__ 1973 1974 ______.. _.. .__
--.___-..
Nov. 2Dec. 15
Dec. 15
Dec. 22
-
+
+
Dec. 29 Jan. 5
Jan. 8Feb. 1
Feb. 1
Feb. 7
Feb. 14
Mar.
Apr.
MayAug.
Sept.
_
+
+
+
_
_
_
_
..--
Therapy Transfer factor Amphoterrcin % intravenous intracisternal Skin tests* Cocci Monilia Blastogenesrs+ Cocci Monilia Antibody Serum Cerebrosprnal
Unrelated
+
+
donor
Related donor ._.~_____
4
___-.
.-... _------~-._---)
.W
* 0 13 0.6-I .1 3.8-10.7 1:3X-1:160 I:20
fluid Cerebrospinal
+
7 17
4 ND
0 11
0 ND
13.7 8.1
5.3 8.5
2.6 11.8
1.9 7.4
0 12 0.9-I .5 6.7-9.9
3 15
8 ND
17 12
>15 .15
315 >I5
>I5 >15
>15 >15 10.9 17.2
8.9 12.0
11.2 17.7
5.5 10.8
5.7-l 1.4 9.4-16.8
13.3 15.2
9.0-l 5.1 8.5-14.7
1:128 1:8
I:128 1:8
ND ND
1:128 I:8
ND ND
1:128 1:8
ND ND
ND ND
I:32 0
1:16 0
1 :16 0
-
_
ND
-
ND
_
ND
ND
_
-
_
I:8 0 -
culture NOTE: Rules indicate duration of therapy. ND = not done. Skin test reactions to coccidioidin 1 :lO for the donors were: unrelated donor 37 mm induration, related donor 43 mm and to monilia 1 :I00 were: unrelated donor 21 mm, related donor 17 mm. *Skin tests were applied 72 hours after the dose of transfer factor was given and were measured as millimeters of induration at 48 hours. + Expressed as a blastoqenic index: cpm of ‘H-thymidine uptake for lymphocytes incubated with antigen divided by uptake following incubation
with medium
alone.
coccidioidomycosis at age 13. He had remained skin test positive and when intradermally tested with soluble coccidioidin reacted with 8 mm of indurationusing a 1:1000 dilution and 37 mm of indurationwith a 1:lO dilution. These skin tests were recorded at 48 hours. The patient was given 1 unit (equivalent to 1 X log lymphocytes) of transfer factor subcutaneously at weekly intervals for four consecutive weeks (Table I). Three days after receiving the first unit she was retested, at which time the skin test response to 1: IO coccidioidin demonstrated induration of 6 by 7 mm. In vitro lymphocyte blastogenic responses increased from a blastogenic index of 1.07 to 13.7 at this time. These responses, however, were transient, and both in vivo and in vitro measurements reverted to negative despite the administration of additional transfer #actor. The patient deteriorated neurologically as a result of obstructive hydrocephalus and a ventriculoperitoneal shunt was placed. Three months after her admission, it was learned that her father had had pulmonary coccidioidomycosis at age 13. He was strongly positive to a 1:iO dilution of coccidioidin intradermally reacting with 43 mm of induration. Dialyzable transfer factor was prepared from the patient’s father as previously described and the patient was given 1 unit subcutaneously each week for three consecutive weeks. The coccidioidin skin tests after the first unit were positive at 8 mm of induration and following the third unit increased to 17 mm of induration. Her blastogenic index after administration of the first unit increased from 1.45 to 8.9. Both the skin test and the blastogenic index remained positive over the eight months they were followed. Following
therapy with transfer
factor from her father, the patient experienced gradual but progressive improvement over four months. Her condition then stabilized
and no further
significant
improvement
curred over an additional 15 months of observation.
oc-
COMMENTS The more severe prognosis for coccidioidomycosis in the pregnant patient is most likely attributable to the known transient depression of cell mediated immunity demonstrated during gestation [8]; the risk of dissemination then increases to an estimated 20 per cent, 100 times that of the nonpregnant white woman and three times that of the nonpregnant black [2]. A majority of reported cases have occurred during the third trimester [l-3]. The exact mechanism of this maternal response remains poorly understood but is apparently directed to protect the developing fetus from rejection. Such tolerance during pregnancy may carry over to newly introduced foreign antigens such as fungal antigens with resultant proliferation of suppressor T cells and subsequent depression of cell mediated immune reactions to the invading organisms [9]. Accepting this hypothesis, the role of immunotherapeutic products like transfer factor can better be explained. Another suggested mechanism is the elaboration of an inhibitory factor into the serum during gestation which depresses cellular immunity [lo]; in the present studies, no such factor could be demonstrated since C. immitis blastogenie assays for known positive controls were not decreased in the presence of the patient’s serum, nor were her responses any different when comparing AB positive serum or fetal calf serum to autologous serum as the medium supplement. The present case illustrates the associations between pregnancy, disseminated coccidioidomycosis and defects in cellular immunity. In this patient the defect
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ET AL
appeared specific to C. immitis since in vivo and in vitro reactivity to other fungal, viral and bacterial antigens as well as nonspecific in vitro stimulation with mitogens was normal. A therapeutic effect was achieved when coccipositive transfer factor from a related donor was combined with amphotericin B. No apparent clinical response and only transient conversbn of cellular immune responses was seen with amphoteiicin B and coccipositive transfer factor from the un&lated donor during a four month period of observation:” It is of course not certain that the clinical benefit was related primarily to the second series of transfer factor but most evidence, as observed,‘suggests this. This question then remains whether the fact that the transfer factor donor was related to the recipient was a significant factor in success of therapy. The skin test and in vitro responses of the two transfer factor donors were comparable and therefore indicate that potency of transfer factor as judged by donor responses alone could not account for the differences in efficacy. A number of clinical reports have attested to the efficacy of transfer factor for severe coccidioidomycosis
as well as for other infectious processes such as chronic mucocutaneous candidiasis, and available information for transfer factor therapy has been summarized previously [ 111. A recent review from the Coccidioidomycosis Cooperative Treatment Group (CCTG) represents the largest series of patients, 42, treated with transfer factor for coccidioidomycosis [ 121 and the present case was it$uded in this study. Clinical and immunologic responses were variable for the 42 patients, but data did suggest clinical efficacy of transfer factor. More definitive information hopefully will be obtained by the CCTG in a controlled prospective trial of placebo transfer factor versus coccidioidomycosis positive transfer factor currently in progress. Finally, in studies employing transfer factor as immunotherapy for patients with osteogenic sarcoma, donors with similar environmental exposure appear preferable, and these are often relatives [ 131. The present report suggests that both the environmental exposure to the agent, in this case C. immitis, as well as the donor being related to the recipient of transfer factor may be of importance for efficacy of transfer factor therapy.
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Smale LE, Waechter KG: Dissemination of coccidioidomycosis in pregnancy. Am J Obstet Gynecol 107: 356, 1970. Harris RE: Coccidioidomycosis complicating pregnancy. Report of 3 cases and review of the literature. Obstet Gynecol28: 401, 1966. Hadsall FJ, Acquarelli MJ: Disseminated coccidioidomycosis presenting as facial granulomas in pregnancy: a report of two cases and a review of the literature. The Laryngoscope 83: 51, 1973. Pappagianis D: Personal communication, 1975. Steele RW, Cannady PB Jr, Moore WC Jr, Gentry LO: Skin test and blastogenic responses to sporothrix schenckii. J Clin Invest 57: 156, 1976. Steele RW, Hensen SA, Vincent MM, Fuccillo DA, Bellanti JA: A 51Cr microassay technique for cell-mediated immunity to viruses. J lmmunol 110: 1502, 1973. Lawrence HS: Transfer factor. Adv lmmunol 11: 195. 1969.
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Thong YH, Steele RW, Vincent MM, Hensen SA. Bellanti JA: Impaired in-vitro cell-mediated immunity to rubella virus during pregnancy. N Engl J Med 289: 604, 1973. Kantor FS: Infection, anergy and cell-mediated immunity. N Engl J Med 292: 629, 1975. Ceppellini R, Bonnard GD, Coppo F: Mixed leukocyte cultures and HL-A antigens. I. Reactivity of young fetuses, newborns and mothers at delivery. Transplant Proc 3: 58, 197 1. Kirkpatrick CH, Gallin JI: Treatment of infectious and neoplastic diseases with transfer factor. Oncology 29: 46, 1974. Graybill JR: Transfer factor in coccidioidomycosis. Clin Res 23: 304. 1975. Levin AS, Byers VS, Fudenberg HH, Wybran J, Hackett AJ, Johnston JO, Spitler LE: Osteogenic sarcoma: immunologic parameters before and during immunotherapy with tumor-specific transfer factor. J Clin Invest 55: 487, 1975.
