mycoses 31, No. 3 (1988) C~~ptococcus neoformans in Quebec 123

mycoses 31 (3) 123-128 . accepted/angenommen: Dezember 21, 1987 . 0 Grosse Verlag Berlin 1988

I Cryptococcus neoformans in Qukbec (1985-1986) I Cryptococcus neoformans in Quebec (1985-1986)

G. St-Germain', P. Auger' and C. Lemieux3 I Laboratoire de mycologie, Laboratoire de sante publique du Quebec * Microbiologie et immunologie, Universite de Montreal, Montreal, Quebec

Dkpartement de microbiologie, HBpital Notre-Dame, Montreal, Quebec

Key words: Cryptococcus neoformans - var. gattii - cryptococcosis - AIDS Schliisselworter: Cryptococcus neoformans - var. gattii - Cryptococcose - AIDS

Summary :Thirty three strains of Cryprococcus neoformans isolated from 33 patients were referred for diagnostic purposes to the Laboratoire de santk publique du QuCbec during 1985-86. The case histories of these patients were reviewed and the strains were examined for their morphological and biochem- ical characteristics as well as their susceptibility to 4 antifungal agents. The predominant sites for recovery of the organism were the respiratory tract (15), blood (14) and CSF (13). Twenty eightofthese33 patients hadadiagnosis of cryptococcosis. Among these 28 patients, meningeal involvement was present in 13 cases and a serious underlying disease was present in 22 cases. Eleven patients had AIDS. The morphological and biochemical characteristics of all 33 strains corresponded in most aspects to the description of the species except for one strain which was urease negative. Typing of strains revealed that one isolate from an AIDS patient belonged to variety garrii of Cryptococ- cus neoformans. In vitro susceptibility testing indicated that all strains were susceptible to amphotericin B, 5-fluorocytosine, ketocona- zole and itraconazole.

Zusammenfassung: DreiunddreiDig Crypto- coccus neoformansStamme, isoliert aus 33 Patienten, wurden in den Jahren 1985 und 1986 zur Labordiagnostik an das Laboratoire de santk publique du Qukbec eingesandt. Die

Krankengeschichten wurden analysiert und die Stamme nach morphologischen und biochemi- schen Kriterien charakterisiert und auf ihre Empfindlichkeit fur 4 Antimykotika gepruft. Die Pilze wurden vorzugsweise aus dem Respirationstrakt (15), Blut (14) und Liquor (13) isoliert. Bei 28 Patienten bestand eine Cryptococcose. Von diesen 28 Patienten zeigten 13 reine meningeale Beteiligung, und 22 hatten eine ernste Grundkrankheit. Elf Patienten hatten AIDS. Die morphologischen und biochemischen Merkmale aller 33 Stamme stimmten in den meisten Aspekten mit der Species-Beschreibung uberein mit Ausnahme eines Stammes, der Urease-negativ war. Die Stammtypisierung ergab, daO ein Isolat von einem AIDSPatienten zur Varietat garrii von Cryptococcus neoformans gehorte. Die In vitro- Testung ergab, daO alle Stamme fur Amphoteri- cin B, SFluorcytosin, Ketoconazol und Itraconazol empfindlich waren.

Cryptococcosis is a chronic, subacute or acute pulmonary, meningeal or systemic mycosis (1 1). The etiologic agent, Cryptococ- cus neoformans, is an ubiquitous encapsulated yeast most frequently associated with pigeon droppings. 'With the increased use of immunosuppressive agents, the prolonged survival of severely immunocompromised patients and the ongoing AIDS epidemic, the number of opportunistic infections caused by

124 G. St-Gerrnain et al. rnycoses 31, No. 3 (1988)

fungi is increasing (9). C. neoformans is now considered the most common significant fungus cultured from clinical specimens next to Candida (2, 11, 13). The present report describes the morphological and biochemical characteristics, as well as the susceptibility to 4 antifungal agents of 33 strains of C. neoformans isolated during a 2 year period in the province of Quebec. It also provides demographic and clinical data concerning the patients from which these strains were isolated.

Material and Methods Clinical data and strains

Clinical data were obtained by the use of a questionnaire filled-in by the physicians who provided the strains. Thirty-three clinical isolates of C. neoformans were studied. They had been isolated from 33 patients in 14 hospitals from January 1985 to December 1986. Four authenticated serotype strains of C. neoformans from the American Type Culture Collection (ATCC # 28957, 32608, 34878, 38871) were included as controls.

Identification procedure

Strains for all physiological tests were grown on Sabouraud dextrose agar for 24-48 h at 30°C. Cells from these cultures were examined for the presence of a capsule using India ink. Absence of pseudomycelium was verified on corn meal agar with polysorbate 80. Assimilation of carbon compounds was performed with the API 20C identification system as well as Wickerham broths (17). Growthat 37"C(17), fermentation (17), urease production (17- 18), phenoloxydase produc- tion (14), nitrate reductase activity (17) and arbutin splitting (17) were also tested. Biochemical serogrouping of all strains was performed on Kwon-Chung's L-canavanine- glycine-bromothymol blue agar (7).

Antifungal susceptibility testing

In vitro susceptibility studies were performed with amphotericin B (Fungizone, Squibb Canada, Montrkal, Qukbec), 5-fluorocytosine

(5-FC) (Hoffmann-LaRoche, Brampton, Ontario), ketoconazole and itraconazole (Janssen Pharmaceutica, Mississauga, Ontario) using an agar dilution technique (8) and a Cathra replicator equipped with 3 mm needles. The azoles were dissolved in dimethyl sulfoxide and tested on Kimmig agar (3).

Results C. neoformans was isolated from 33 patients, whose ages ranged from 29 to 86 years (Fig. 1). Twenty seven of them were male. Twelve male patients in the 30 to 39 year age interval constituted the most important group among all age groups; eight of these 12 patients had AIDS.

Clinical specimens most frequently found to be positive for C. neoformans included respiratory tract specimens, blood and CSF. Frequency of sites with positive cultures is reported in Table 1.

Twenty eight of our 33 patients were diagnosed as having cryptococcosis and 46% ofthem had meningitis (Table 2). In the other 5 patients, C. neoformans was isolated from the respiratory tract and an abdominal abscess associated with carcinoma of the stomach, but clinical or microbiological evidence of dissemination was not observed. These patients were thought to be either colonized or affected by a sub-clinical, transitory infection. None of them were treated with antifungal agents and all survived.

Among the 28 patients with cryptococcosis, the most frequently encountered underlying disease was AIDS (Table 3). Close to 40% of these patients were affected by this disease. Other diseases or predisposing factors encountered were neoplasia and antibacterial and immunosuppressive drug therapy, all of which are known to have adverse effects on host defenses. However, it is noteworthy that 6 patients did not appear to be affected by any underlying disease; even though 3 of them had meningitis, they all responded well to treatment.

The 33 strains of C. neoformans studied were morphologically typical of the species. In all

mycoses 31, No. 3 (1988) Ctyptococcus neoformans in Quebec 125

MALES FEMALES 12

12

2 9 C Q)

0 a 0

Q

.- e

u-

L 6 n E 3 Z

3

n " 0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89

Age

Fig. 1: Distribution of 33 patients by age group and sex.

Table 1: Sites from which Cryptococcus neoformans was recovered on primary isolation in 33 patients

Site Number

Blood CSF Bronchial washings Lung biopsy Skin Urine Synovial fluid Pus from abdominal abscess

14 13

9 6 3 3 1 1

Table 2: Clinical manifestations attributed to Cryptococcus neoformans in 28 patients

Oiaanosis No. of patients

Meningitis Primary fungemia Pneumonia Cellulitis Synovitis Cryptococcoma

13 6 4 3 1 1

Total 28

cases, yeasts were spherical, encapsulated and did not produce any pseudomycelium. All strains grew at 37°C. Fermentation tests were negative. The results for 34 biochemical characters are recorded in Table 4. One strain isolated from an AIDS patient was found to belong to the gattii variety and was confirmed as a serotype B by the National Institutes of Health in Bethesda. In vitro susceptibility tests indicated that all strains were susceptible to the 4 antifungal drugs tested (Table 5).

Discussion

Cryptococcosis is one of the infectious complications of AIDS (5). In our study nearly 40% of the cases involved patients with AIDS. Even though cryptococcosis in general is reported more often in males than in females (1 l), the high number of male patients in this study is possibly a consequence of the predominant number of males versus females affected by AIDS in Quebec during those 2

126 G. St-Germain et al. mycoses 31, No. 3 (1988)

Table 3: Underlying diseases in 28 patients with a diagnosis of cryptococcosis

Disease

AIDS Leukemia Cirrhosis Immunosuppression Leukopenia Lymphoma Mycosis fungoides Pulmonary neoplasia Sarcoidosis None

Total

No. of patients

1 1 (39,3)% 3 (10.7) 2 (7.1) 1 (3.6) 1 (3.6) 1 (3.6) 1 (3.6) 1 (3.6) 1 (3,6) 6 (21.3)

28 (loo)% ~~~

Table 4: Frequency of occurence of test characteristics among 33 strains of Cryptococcus neoformans

Test name Number Number Percent positive negative positive

0-glucose 0-galactose L-sorbose sucrose maltose cellobiose trehalose lactose melibiose raffinose melezitose starch 0-xylose L-arabinose 0-arabinose 0-ribose L-rhamnose glycerol erythritol ribitol galactitol 0-mannitol 0-glucitol salicine OL-lactic acid succinic acid citric acid inositol inuline methyl-a-0- glucoside arbutin nitrate phenoloxydase urease

33 33 1 1 33 33 33 33 0 0 28 33 33 33 32 33 30 32 2 26 31 33 33 33 33 8 14 24 33 26

33 20 0 33 32

0 0 22 0 0 0 0 33 33 5 0 0 0 1 0 3 1 31 7 2 0 0 0 0 25 19 9 0 7

0 13 33 0 1

100.0 100.0 33.3 100.0 100.0 100.0 100.0 0.0 0.0 84.9 100.0 100.0 100.0 97.0 100.0 90.9 97.0 6.1 78.8 93.9 100.0 100.0 100.0 100.0 24.3 42.4 72.7 100.0 78.8

100.0 60.6 0.0

100.0 97.0

Table 5 In Vitro susceptibility of 33 isolates of Cryptococcus neoformans

MIC (mg/L) Antifungal agent Range Mean

Amphotericin B 0,005 -0.02 0.01 5-Fluorocytosine 0,lO -1.56 0.36 Ketoconazole 0.10 -0.73 0.30 ltraconazole 0.0125-0.20 0.05

years. Cryptococcosis is most often considered to be an opportunistic infection (1 1); however, close to 20% ofour patients with a diagnosis of cryptococcosis did not appear to have any underlying disease. Otherwise, all 28 of these cases present a classic clinical picture with almost half of the patients with meningeal involvement. As far as the frequency of isolation of the yeast from different types of clinical specimens is concerned, our observa- tions are similar to those of Roberts et al. (12).

The biochemical characteristics of our strains are similar to those reported by Schmeding et al. (15) on 97 strains from the American Type Culture Collection. All of our 33 strains grew in 16 carbon sources, D- glucose, D-galactose, sucrose, maltose, cel- lobiose, trehalose, D-melezitose, starch, D- xylose, D-arabinose, galactitol, D-mannitol, D-glucitol, salicine, mesoinositol and methyl- a-D-glucoside. All were phenoloxydase posi- tive. Unexpectedly, one strain was urease negative. This result was obtained repeatedly with both Christensen’s and Zimmer’s methods and was confirmed by the National Reference Center for Human Mycotic Diseases in Alberta and the API Mycology Laboratory in New York. The urease test is often used for the presumptive identification of C. neoformans. To our knowledge, no urease negative strain has ever been reported in the literature. The API 20C system identified all 33 of our isolates correctly even though as many as 21 different biocodes were obtained.

Strains of C. neoforrnans can be further characterized as belonging to 2 varieties; variety neoformans(serotypes A, D, AD) which is known to be distributed world-wide and

mycoses 31, No. 3 (1988) Cryptococcus neoformans in Quebec 127

associated to pigeon excreta, and variety gattii (serotypes B, C) which is prevalent in the tropical and subtropical areas of the world (6). The biochemical serogrouping of our strains indicated that one of them belonged to variety gattii. This is the second report of a gattii strain isolated from an AIDS patient (4), previous reports indicating that until recently, C. neoformans var. neoformans was the sole cause of cryptococcosis in these patients (10, 16).

Nineteen of our 28 patients with cryptococ- cosis were treated with a combination of amphotericin B and 5-FC. Two other patients were treated with amphotericin B or 5-FC alone. A pre-auricular ulcer in one patient was treated successfully with ketoconazole alone. The other 6 patients were not treated either because they were in terminal phase of their underlying disease or because the diagnosis was established post-mortem. Susceptibility studies indicated sensitivity of all 33 strains to amphotericin B and 5-FC. Yeasts are in general susceptible to amphotericin B (1 1) and in vitro testing is usually not useful except in cases where the patient is not responding to treatment. However resistance to 5-FC has been reported especially after treatment has been instituted (1, 11). Our strains were isolated before treatment and none showed any sign of resistance to this drug. Both ketoconazole and itraconazole significantly inhibited growth of all strains in our test system with MIC's < 0,3 mg/L. However, azoles in general are seldom used for the treatment ofcryptococcosis in humans and are still under evaluation. A combination of amphotericin B and 5-FC remains the treatment of choice (1 1) especially when the central nervous system is involved.

In conclusion, serious cases of cryptococ- cosis are encountered in Quebec every year. As in many countries around the world, an important number of our cases are related to the AIDS epidemic. This report is the first of its kind produced in Canada. All in all, it indicates that cases of cryptococcosis observed here are similar in characteristics to those that are observed elsewhere in North America. The biochemical characteristics of our trains are

typical of the species with the exception of one urease negative strain. No in vitro resistance was found to amphotericin B or 5-FC. We report the second known case of cryptococ- cosis in an AIDS patient caused by variety gattii of C. neoformans.

Acknowledgement: The authors wish to acknowledge the technical assistance of Ms. M. Guertin and Ms. J. Jacques. We would also like to thank Dr. K. J. Kwon Chung for the serotyping of the gattii isolate.

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11. Rippon, J. W.(1982): Medical Mycology. Philadelphia: W. B. Saunders. pp. 532-558.

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14. Salkin, I. F. (1979): Further simplification of the Guizotia abyssinica seed medium for identification of Cryptococcus neoformans and Cryptococcus bacillis- pora. Can. J. Microbiol. 25, 11 16-1 118.

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Address: Mr. Guy St-Germain, Laboratoire de Sante Publique du QuBbec, 20045 chemin Ste-Marie, Sainte-Anne-de-Bellevue, Quebec H9X 3R5, Canada.

C ongress-Calendar KongreB kalender

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