CD4 lymphocyte count in HIV positive persons exposed to cryptococcus neo formans

Zbl. Bakt. 283, 127-135 (1995) © Gustav Fischer Verlag, Stuttga rt · Jena . New York

CD4 Lymphocyte Count in HIV-positive Persons Exposed to Cryptococcus neoformans K. ARASTEHI, M. L'AGE 1 , U. FUTH 2 , G . GROSSE l, and F . STAIB 'II. Innere Klinik (Leiter: Prof. Dr. M. L'age), Abteilung fur Labor atoriumsmedizin (Leiter: Prof. Dr. R. Averdunk), 3 Institut fur Pathologie (Leiter: Prof. Dr. F. Niedobitek) Auguste-Viktoria -Krankenhaus, 0-12157 Berlin/Germany 1

2

Received Ma y 24, 1995 . Accepted July 20,1995

Summary A report is presented on four HIV-positive homo sexual men examined after several months of exposure during cleaning of a flat from masses of pigeon droppings heavily colonized by Cryptococcus neoformans . Only one out of the four persons, with a CD4 lymphocyte count of 50/[tL, fell sick from systemic cryptococcosis, but not the others, with CD4 lymphocyte counts of 180, 250, and 630/[tL, respectively; they remained clinically and mycologically inconspicuous and free from C. neoformans. Open questions in view of the epidemiology of opportunistic pathogens in AIDS are discussed with regard to the CD4 cell count as a parameter indicating a predisposition for cryptococcosis as an airborne AIDS-defining opportunistic infection. This has been confirmed by specific cultural diagno sis of the agent in both the environment and the patient. Already in 1987/88, the probable source of infection had been the subject of epidemiological studies on C. neoformans in Berlin.

Introduction CD4 lymphocytes are thought to be an essential part of th e defense mechanisms of the human body against Cryptococcus neoformans (11,12,18,19). As a consequence of disturbed T-cell-mediated immunity in HIV infection, the epidemiological and pathogenetic significance of opportunistic pathogens, among them the basidiomycetic yeast C. neoformans, is placed in the focus of clinical control. Examination for clini cal and biological parameters of HIV infection is of foremost sign ificance. As a guide, the absolute count of CD4 T cells and the demonstration of HIV antigens (e.g. p24) is thought to be expedient (17). At a corresponding constellation of such parameters, there is an interest in examinations for th e various causative agents of AIDS-defining diseases (1,2,6,10,14-17,23,24).

* Former Chief Mycology Unit, Robert Koch Institute , Berlin.

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C. neoformans is a frequent opportunistic pathogen in AIDS patients (6, 7, 14, 15, 21,25) . The course of the corresponding airborne infection is known (9,21). If diagnozed early enough, an effective therapy is possible (6, 15, 16,21,25). This systemic infection is combined with a C. neoformans-specific production of phagocytosis-inhibiting capsular polysaccharides supposed to be responsible for the acute course of cryptococcosis in AIDS (3, 4, 14,21). The specific cultural' detectability of C. neoformans makes it possible to examine not only the immediate environment of risk persons for habitats of C. neoformans (Fig. 3), but also for following the course of infection in man quantitatively (5, 8, 15,21,23) (Fig. 1). Among others, the determination of parameters in view of a specific antimycotic prophylaxis should be an aim of such environmental and clinical examinations (24). In the following, some clinical and epidemiological observations made in four HIVpositive homosexuals after their exposure to C. neoformans habitats will be reported. Patients and Methods Four HIV-positive homosexuals were cleaning a flat from masses of pigeon droppings over several months in 1994 . One out of them fell sick from cryptococcosis . On the basis of this history, an exposure to airborne cryptococci from pigeon droppings was assumed. Therefore, the patient's home and neighbourhood were examined for habitats of C. neoformans . Beside the four HIV-positive persons' questionable exposure to C. neoformans, their clinical and immune status represented by their CD4 counts was of interest . 1. Cryptococcosis patient Medical History

A thirty-year-old homosexual man who had suffered from fever (over 40 o q , severe dysbasia for one week and double images was admitted to the hospital. A HIV infection had been diagnosed in 1989 . SinceJuly 1994, based on a diagnosed Candida esophagitis, he had been categorised as C3, following the CDC staging of 1993. Esophagitis was treated by administration of fluconazole 200 mg/d over 4 weeks. To prevent PcP, he inhaled 300 mg pentamidine at monthly intervals. Up to now, he has not received antiretroviral treatment. Clinical status

The patient was in a reduced general state of health . Physical examination revealed a significant organic brain syndrome with disturbance of awakeness as well as an emphatic brachy -cervicalleft side symptomatology with monotonal speech impediments and gaze coordination disturbances as in the case of strabismus convergens. Further findings: Epididymitis right hand side. Other clinical examinations did not produce further pathological findings. Clinical-chemical and microbiological findings

Mild anemia HB 10.9 g/dL, clear increase of HBDH with 262 U/L, CD4 lymphocyte count 50/~L (Norm 650-1250/~L), acid-dissociated p24-AG 19 pg/mL. C. neojormans antigen in serum 1 : 2560; for further C. neoformans findings see Table 1. Lues serology negative, toxoplasmosis-IgG (ELISA) positive, CSF: Proteins 69.0 mg/dl., no increase of cell count . peR of Mycobacterium tuberculosis and M . avium intracellulare

negative, cultures of Mycobacterium spp. negative after 6 weeks.

C. neojormans and CD4 lymphocyte count

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Important supplementary examinations Chest X-ray: Inconspicuous. NMR tomography: Multiple foci with marginal enhancement and central contrast medium resorption both sides supra and infra tentorial. Marked formation of perifocal edema. A contact with the CSF system at the focus side was seen in each case, so that the differential diagnosis besides a cerebral toxoplasmosis above all could have been a cryptococcal meningitis. Cranial computed tomography: Init ially, no clear diagnosis after 14 da ys by size-consta nt foci. Cerebral toxoplasmosis wa s of foremost importance in differential diagnosis. Treatment and clinical course Altogether, cryptococcosis was treated for 6 weeks with a triple combination consisting of amphotericin B (0.5 mg/kg/d i. v.), flucytosine (150 mg/kg/d i. v.), and fluconazole (400 mg/d i. v.). Because of a delayed success of clinical ther apy, the changed expr ession of the imaging cranial diagn ostics and serological positi vity of the toxoplasmosis-IgG marker after 14 days, a toxoplasmosis therapy was added. Treatment followed in the usual wa y with pyrimethamine (150 mg/d ), sulfadiazine (6 g/d ), and folic acid (30 mg/d) . Based on an intercurrent toxic hepatitis with increas e of the transaminases to over 1000 U/l, therapy was interrupted until a clear decrease of transaminases could be seen. Finally, itraconazole 200 mg/d was given as a cryptococcosis maintenance therapy, as well as atovaquone 4 X 750 mg/d for continued cerebral toxoplasmosis prophylaxis. After a tot al of 9 weeks of hospitalization, the patient was discha rged in a stable condition and since then has received outpatient treatment. 2. CD4 lymphocyte determination CD41ymphocyte counting (Norm 650-1250/Ill) was performed with the aid of an FACScan flow cytometer and reagents from Becton Dickinson Immunocytom etry Systems, San Jose, California 955113 (Table 1) (20). 3. Mycological examinations Clinical specimens : The mycological examination of specim ens for C. neoformans was based on light microscopy of native prep arations from the mate rials and on culturing (21, 23) . Specimens examined by culture con sisted of blood, sputum, cerebros pina l fluid (CSF), urine , seminal fluid, and prostatic secretion . The cultural examinations were performed with the help of Staib agar (syn. Gui zotia abyssinica creatinine agar, bird seed agar), on wh ich coloni es of C. neoformans produced the typical brown colour effect (BCE) with in 35 d/26 DC (Fig. 1) (21) . All strains showing the BCE wer e controlled for identity using mycological standard methods (including thei r varietal status) (14, 2 1, 22). For the serodiagnosis of cryptococcosis, the Immy latex Cryptococca l Antigen System was used (21 ). Pigeon droppings: From the patient's home and neighbourhood, sampl es were taken in major amo unts (22 ). From these sam ples, ca. 1-5 g each were suspended in 5 mL of sterile distilled water and streaked o n plates with Staib agar, supplemented with penicillin G (20 UI mL), streptomycin sulfate (40 U/ml), and biphen yl (0.01 % and 0.1 % ). The currently used formula of Staib agar has been described elsewhere (21). On account of the high CFU counts of C. neoformans in the mat erial examined, dilutions of the suspensions were needed . Becau se the bird droppings were highly contaminated with spores of hyphomycetes, biphenyl supplementation of Staib agar was necessa ry to inhibit the growth of hyphom ycetes (2 1) (Fig. 3). Since biphenyl also produces a certain inhibition of C. neoformans growth, Staib agar plates without and with both concentrations of biphenyl, i. e. 0.0 1 % and 0.1 %, were used for each examination of samples. The inoculated plate s were incubated at 26 °C for up to 14 days . From the 2nd da y on, the plate s were observed dail y for the growth of yeast-like fungi to detect colonies showing the BCE being typical of C. neoformans (Fig. 3).

1988

G. 29y.

0

0

0

0

+

0

0

+

o

+

Expos. to pigeon dropp. MaySep.1994

0

0

0

+

+

+

200 mg/d + 4 weeks Ju ly'94

AZT TMPI Fluco SMX nazole

Therapy

= e. neoformans culture positive or exposed to pigeon droppings = e. neoformans culture or antigen nega tive n.d. = not detectable

Signs and abbrevations:

6

250 n.d. Oct. '94

630 n.d. Oct. '94

19 50 Nov. ' 94

CD4 cell p24-AG counr/ul, pg/mL

B. 1987 Pneumonia, 180 32y. oral thrush Jan. '95 '93194 0

0

D. 42y.

0

1988

1989 Thrush 1994

E. 30y.

0

HIV- Manifepos. stations since observed

Pat.

p24-AG AZT TMP SMX

Dec . '94

Feb . '95

Dec . '94

Feb . '95

Dec . '94

Oct'! + Nov. '94 Febr. '95

0

0

0

+

Sputum

0

0

0

0

}

Urine

= HIV antigen = azidothymidine = trimethoprim = sulfamethoxazo le

0

Blood CSF

Culture

Serology

+ = therapy

o = no therapy

0

0

0

0

0

0

1:2560

Sper- Prost. Antigen rna seer.

e. neoformans diagnosis

Table 1. Histories, results and therapy in 4 HIV-positive persons after joint exposure to Cryptococcus neoformans airborne in dust of pigeon droppings not exposed to weather conditions

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0

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C. neoformans and CD4 lymphocyte count

131

Results

1. Th e cryptococcosis patient: Clinical and epidemiological examinations The cultural detection of C. neoformans var. neoformans in blood and sputum (Table 1) (Fig. 1) in connection with a C. neoformans antigen titre of 1 : 2560 in serum proved the presence of the secondary stage of the C. neoformans infection with a duration of 3-4 months (21). This means that the Candida esophagitis (Table 1) was present at the time of the clinicall y non-recognized on set of the C. neoformans infect ion. Presumably, the treatment of Candida esophagitis with fluconazole (200 mg/d) in Jul y '94 over 4 weeks caused the protracted onset of cryptococcosis (Table 1). Also, the question arise s if in October ' 94, the missing cultural detection of C. neoformans in CSF and urine had been a con sequence of the acute therapy consisting in the administration of amphote ricin B, fluctyrosine, and fluconazole, starting 2 and 4 da ys, respectively, before the mycological exam inations were performed. Th e flat of the cryptococcosis pati ent was at the back of a multiple-dwelling house adjacent to a church whose tower rooms were found in 198 7/88 to be mas sively contaminated with pigeon droppings (not exposed to weather conditions) heavil y colonized by C. neojormans var. neoformans (22). In 1994, nets were installed to prevent an intrusion of pigeon s. Thus, the se pigeons looked for nest ing places in the adjacent hou ses where the cryptococcosis patient had rented a flat in Apr il 1994. In December '94, at th e time of the inspection of the renovated flat by the author (ES.), there wa s still a room in the bu ilding from which the pigeon droppings had not yet been removed

I

-

Fig. 1. Cultural examination of sputum of pat ient E. for C. neoformans with the aid of Staib agar (after 8 days/26°C) (Table 1). Because of their intense BCE (arrow), the colonies of C. neoformans may be easily differentiated from the non-pigmented colonies of the various species of yeasts, e. g. of Candida spp. Note : In the black-and-white photogr aph, the intense BCE appears in black. Diameter of Petri dish 80 mm.

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K. Arasteh et al.

Fig.2. Accumulations of pigeon droppings not exposed to weather conditions. This garbage chute room belongs to the building in which the cryptococcosis patient's flat is situated. Cryptococcosis was diagnosed in patient E. 3-4 months after cleaning of such rooms (s. Figs. 1, 3).

(Fig. 2). This was a former garbage chute room connected to a staircase of the build ing. In these pigeon droppings, > 2 X 106 CFU of C. neoformans var. neoformans/g were found . Beside C. neoformans, there was heavy growth of mucoraceae, Rhizopus spp . and among these, Rh . oryzae (Fig. 3).

2. Clinical and mycological examination of HI V-positive persons after exposure to pigeon droppings colonized by C. neoformans var. neoformans As shown in Table 1, the cryptococcosis patient had a CD4 count of 50/~L (Norm

650-1250fl,tL) while the others had counts of 180, 250, and 630fl,tL. Mycological as well as clinical examinations of the 3 Hl V-positive friends of the cryptococcosis patient revealed that they were free from symptoms and negative for C. neoformans (Table 1). So far (after an observation period of 6 months), these 3 HIV-positive persons have remained clinically symptom-free.

Discussion The example of the C. neoformans infection in a HIV-positive man has shown which epidemiological examinations, in addition to clinical care, may come under the clinician's responsibility, if dealing with Hl'V-positive persons.

C. neoformans and CD4 lymphocyte count

133

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Fig. 3. Cultural examination of pigeon droppings (s. Fig. 2) for C. neojo rmans with the help of Staib agar supplemented with penicillin G (20 U/mL), streptomycin sulfate (40 U/mL), and biphenyl 0.01 % after 5 days/26°C. Because of the high count of > 2 X 106 CFU of C. neo(ormans/g, high dilutions of the suspensions of the pigeon droppings were necessary. The colonies of C. neoformans with an intense BCE (ar row) may be easily recognized, appearing black in the black-and-white photograph. Note: The fine growth of mucorace ae over the whole surface of the plate demonstrates that there has not been a complete inhibition of growth of mucoraceae at a biphenyl concentration of 0.0 1%.

Exposure A C. neoformans antigen titre of 1 : 2560 found in the serum of the HIV-positive patient at the time of his admission to the hospital suggested a course of infecti on with a duration of approximately 3-4 months (21). A search for the time of exposure revealed that the probable onset of the infection wa s during a period wh en the newly rented flat was cleaned from huge accumulations of pigeon droppings by the four HIVpositive persons. It mu st be ass umed that at the time of cleaning, C. neoformans had colonized the se rooms as massivel y as th e still uncleaned form er ga rbage chute room (the CFU count of C. neoformans var. neoformans in the accumulated masses of pigeon droppings was > 2 X 10b/g!) (Figs. 2, 3). Epid emiologically, these observat ion s raised interest because in 198 7/88, the tower of th e adjacent church was the only on e out of 14 towers polluted by pigeon droppings th at was found to have been colonized by C. neoformans (22 ). Already, at that time and by this ob servation, it was demonstrated and discussed that pigeon droppings are not uniforml y colonized by C. neoformans as often erroneously stated, but to a small percentage only. From this statement, the still valid question arises how far the resulting relatively small percentage of C. neo-

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formans infections among AIDS patients (3-5%) may be attributed to such a limited exposure to C. neoformans habitats (22). CD4 lymphocyte count

A CD4lymphocyte count of < 100-200/[tL means a predisposition to cryptococcosis as an AIDS-defining disease (13, 14,24) as in the case of mucocutaneous candidiasis (thrush) (6) (Table 1). Such criteria may be applied to the observation presented that out of four HIV-positive men having been exposed to pigeon droppings colonized by C. neoformans, only the one with a CD4 lymphocyte count of 50/[tL fell sick from cryptococcosis, but not the other ones with CD4 lymphocyte counts of 180, 250, and 630/[tL. Correspondingly, their clinically and mycologically negative findings meant a normal defense and elimination of the inhaled cryptococci. Also by examination of the seminal fluid of two of them, it could be demonstrated that due to the inhalation of C. neoformans (Blastospores or basidiospores) by the two persons with CD4 lymphocyte counts of 250 and 630/[tL, respectively, no latent clinically inconspicuous infection of the prostate was found . This was of interest because the prostate may be a symptomless niche colonized by C. neoformans (7, 15, 16,21,23) (Table 1). In an exemplary case of an AIDS patient in the secondary stage of cryptococcosis with a CD4 cell count of 10/[tL, a persistence of C. neoformans in the urogenital tract was found so that antimycotic treatment remained ineffective (23). Based on the present findings, the absolute CD4 lymphocyte count of < 100/[tL could be discussed as a parameter indicating the use of an antimycotic prophylaxis (e. g. fluconazole, itraconazole) (24), especially in the case of a risk of exposure to C. neoformans habitats. How far this parameter referring to C. neoformans could also be used for studying the epidemiology of other airborne opportunistic agents, e. g. Pneumocystis carinii, which has not yet been clarified should remain a subject for further clinical and epidemiological investigation (15,21,22). As a consequence of the exposure of HIV-positive persons to a C. neoformans habitat detected as a probable source of infection, claims for damages by persons at risk have to be discussed . An avoidance of such consequences asks for the abolition of the anonymity of risk persons and supervision of C. neoformans habitats like nesting places of pigeons in residental quarters, pet shops, breeding establishments, and zoological gardens not carried out so far by public health services (21,22). References 1. Artigas, l., G. Grosse, and F. Niedohitek : The Central Nervous Systemin AIDS. Springer Verlag Berlin- Heidelberg - New York (1993) 2. Bacellar, H., A. Munoz, D. R. Hoover, ] . P. Phair, D. R. Besley, L. A. Kingsley, and S. H. Vermund: Incidence of clinical AIDS conditions in a cohort of homosexual men with CD4+ cell counts < 100/mm3 • J. Infect. Dis. 170 (1994) 1284-1287 3. Cherniak, R. and J. B. Sundstrom: Polysaccharide antigens of the capsule of Cryptococcus neoformans. Infection and Immunity 62 (5) (1994) 1507-1512 4. Curtis, J. L., G. B. Huffnagle, G. H. Chen, M. L. Warnock, M.R. Gyetko, R. A . McDonald, P.J.Scott, and G. B. Toews: Experimental murine pulmonary cryptococcosis. Differences in pulmonary inflammation and lymphocyte recruitment induced by two encapsulated strains of Cryptococcus neoformans . Lab. Investig. 71(1) (1994) 113-126 5. Denning, D. \v., D. A. Stevens, and J. R. Hamilton: Comparison of Guizotia abyssinica seedextract (bird seed)agar with conventional media for selective identification of Cryp-

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tococcus neoformans in patients with acquired immunodeficiency syndrome. J. Clin . Microbiol. 28 (1990) 2565-2567 6. Dupont, B., D. W. Denning, D. Marriott, A. Sugar, M. A. Viviani, and T. Sirisanthana: M ycoses in AIDS patients. J. Med. Vet. Mycol. 32 (Suppl.) (1994) 65-77 7. Grosse, G.: Die Prostata als Erregerreservoir bei der Cryptococcose. In: Pilzinfektionen bei abwehrgeschwachten Patienten (F. Staib und D. Huhn), p. 29 . Springer Verlag, Berlin - Heidelberg - New York (1991) 8. Grosse, G., F. Niedobitek, M. L'age, and F. Staib: Chronische Lungenkryptokokkose Ein kasuistischer Beitrag zur Diagnostik der Kryptokokkose des Menschen aus pathologisch-anatomischer Sicht. Dtsch. med . Wschr. 106 (1981) 1035-1037 9. Grosse, G., F. Staib und M . Seibold: Zur Pathologie der Cryptococcose bei AIDS. Verh. Dtsch . Ges. Path . 71 (1987) 506 10. Grosse, G., W. Heise und F. Staib: Histoplasmose der Haut als opportunistische Erstinfektion bei AIDS. Dt sch. med . Wschr. 118 (1993) 1555-1560 11. Hill, j. 0.: CD4+ T cells cause multinucleated giant cells to form around Cryptococcus neoformans and confine the yeast within the primary site of infection in the respiratory tract.J. Exper. Medicine 175(6) (1992) 1685-1695 12. Hill.]. O. and K. M. Aguirre: CD4+ T cell-dependent acquired state of immunity that protects the brain against Cryptococcus neoformans. J. Immunol. 152(5) (1994) 2344-2350 13 . Knudsen,]. D. , L.]ensen, T.]ensen, H. Kjersem, T. L. Serensen .]. Stenderup, and C. Pedersen: Cryptococcosis in Denmark 1988-1993. XII ISHAM Congress Adelaide/Australien. Abstracts PO 1.36 (1994) 14. Kwon-Chung, K.]. and]. E. Bennett: Medical Mycology. Lea & Febiger, PhiladelphiaLondon (1992 ) 15. L'age, M., W. Heise, K. Arasteh, j. Skorde, G. Grosse, M. Seibold, and F. Staib : Clinical course and relapse of cryptococcosis. First Internat. Conf. on Cryptococcus and Cr yprococcosis, Nov ember 12-16, Jerusalem/Israel (1989) 16. L'age, M., K. Arasteh, W. Heise, and S. Klatt: Place of flucytosine in the management of cryptococcosis. 2nd Internat. Conf. on Cryptococcus and Cryptococcosis. September 1923, MilanlItaly (1993) 17. Uage-Stehr.]. und E. B. Helm : AIDS und die Vorstadien. Ein Leitfaden fur Praxis und Klinik. Springer Verlag, Berlin - Heidelberg - New York (1993) 18. Levitz, S. M., M. P. Dupont, and E. H. Smail: Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans. Infect. Immun. 62( 1) (1994) 194202 19. Murphy,]. w., M. R. Hidore , and S. C. Wong: Direct interactions of human lymphocytes with the yeast-like org anism, Cryptococcus neoformans. J. Clin . Invest. 91(4) (1993 ) 1553-1566 20. Reichert, T., M. De Bruyere, V. Denise et al.: Lymphocyte subset reference ranges in adult caucasians. Clin . Immunol. Immunopathol. 60 (1991) 190-208 21. Staib, F. : Kryptokokkose bei AIDS aus mykologisch-diagnostischer und -epidemiologischer Sicht. AIDS-For sch. 2 (198 7) 363-382 22 . Staib, F. and M. Heif5enhuber: Cryptococcus neoformans in bird droppings: A hygienicepidemiological challenge. AIDS-Forsch. 4 (1989) 649-655 23 . Staib, F., M. Seibold, and M. L'age: Persistence of Cryptococcus neojormans in seminal fluid and urine under itraconazole treatment. The urogenital tract (prostate) as a niche for Cryptococcus neoformans. M ycoses 33(7/8) (1990) 369-373 24. Stern, j.j. : Cryptococcal meningitis 1994 . Is there really a state of the art? XII ISHAM Congress, Adelaide/Australien. Abstracts S 31.4 ( 1994) 25. Viviani, M. A.: New therapeutic approaches for the treatment of cryptococcosis in AIDS. XII ISHAM Congress, Adelaide/Australien. Abstracts S 3.3 (1994) Dr. med . K. Arasteh, II. Innere Klinik, Auguste-Viktoria-Krankenhaus, Rubensstrafse 125, D-12157 Berlin