J. clin. Path. (1963), 26, 464

Cryptococcal meningitis in Hong KongC. T. HUANG, P. C. WONG, AND C. H. CHAN-TEOH

From the Department ofPathology and Bacteriology, University ofHong Kong

SYNOPSIS The clinical and bacteriological findings in three cases of cryptococcal meningitis whichoccurred in Hong Kong are reported. The bacteriological investigations are discussed.

Littman and Zimmerman (1956) reported that therewere over 300 cases of human cryptococcosis onrecord, yet compared with other systemic mycoseshuman cryptococcosis is still a rare clinical entity.Of all cases of human cryptococcosis reported,cryptococcal meningitis or meningoencephalitis isthe most frequently encountered. The nervoussymptoms and the ease with which the causalorganism can be isolated from the cerebrospinalfluid enable prompt diagnosis. As far as we are awarethere have been eight cases of cryptococcosisreported in China, and the scattered reports havebeen comprehensively reviewed by Cheng (1956).Three more cases in which the patients were alsoChinese appeared in Malaya and Singapore (Pallis,1949; Loh, 1951; Russell and Dean, 1957). Neverthe-less, with a large population and only a dozen or socases reported, it indicates that cryptococcosisamongst the Chinese race must be rare. In contrastto this belief, we have encountered three cases ofcryptococcal meningitis in Hong Kong betweenDecember 1958 and December 1961. All the patientswere Chinese and all died. The clinical records andthe bacteriological investigations of these threecases are presented here to show that the incidence ofcryptococcosis is probably higher than that found inthe medical literature.

CASE REPORTS

CASE 1 A man aged 30 years, occupation unknown, wasadmitted to a private hospital in November 1958 com-plaining of frontal headache, diplopia, insomnia, andrapid loss of weight. Stained smears of cerebrospinalfluid examined at that stage showed acid-fast bacilli. Hewas treated for tuberculosis with streptomycin, P.A.S.and I.N.A.H. for 14 days but there was no significantimprovement and the patient then developed signs ofcerebellar dysfunctioning. He was transferred to theQueen Mary Hospital on 5 December 1958 for furtherinvestigation.Received for publication 14 January 1963

On admission, he was found to be mentally confusedand drowsy. His temperature was 100°F., blood pressure160/100 mm. Hg, and pulse rate 64 per minute. Leftfacial palsy, nystagmus, and bilateral papilloedema werenoted. Ventriculography showed evidence of incompleteposterior fossa block. Tuberculous meningitis withbasal adhesion was suspected, but smears of cerebro-spinal fluid showed numerous yeast-like cells, whichwere subsequently identified as Cryptococcus neoformansby culture. At operation basal arachnoiditis with adhesionwas observed and the adhesion was separated to establishfree passage of cerebrospinal fluid. A portion of themeninges removed for histological examination showedcryptococcosis. Tubercle bacilli were not found by smears,culture, or guinea-pig inoculation. The patient died eightdays after admission. Permission for necropsy wasrefused.CASE 2 A man aged 31 years, single and a tailor byprofession, came to Hong Kong from Shanghai 10 yearsago and was well until two weeks before admission whenhe began to experience attacks of vertigo and severe,persistent headache over the occipital and temporalregions. He had had no fever since the onset of the illness.His parents and 11 brothers and sisters were living withhim but none of them had the same complaint.The patient was admitted to the Nethersole Hospital

on 18 January 1960. On admission, he was mentallyclear, had no neck rigidity, and Kemig's sign was nega-tive. The fundi showed bilateral papilloedema with manysmall haemorrhagic spots. There were no palpable lymphglands. Blood pressure was 130/90 mm. Hg and thepulse rate was 80 per minute.Haemoglobin was 14 g./100 ml.; W.B.C.s 6,700/c. mm.

(74% neutrophils and 26% lymphocytes); E.S.R.4 mm./hr.; the Kahn test was negative.

Cerebrospinal fluid was at a pressure of 250 mm. TheQueckenstedt test showed a quick rise and slow fall andPandy's test was negative. Concentrations of sugar were10 mg./100 ml. and of chloride 460 mg./100 ml. Nocobweb formation or acid-fast bacilli were seen.On the second day after admission and on four occa-

sions subsequently C. neoformans was found in the cere-brospinal fluid either in smears or by culture.

Radiographs of the chest showed tuberculous infilt-ration in the right middle zone; the skull was normal.

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The patient also had an ulcer on the left forearm. Torule out primary foci, the sputum and the scab from theulcer were examined for C. neoformans, which was not,however, found. Mycobacterium tuberculosis was notfound in sputum when examined by smears, culture, andguinea-pig inoculation.The patient was treated with nystatin (500,000 units)

and sulphadiazine (1 g.) given by mouth every four hours.Subjectively he felt better, but his condition suddenlydeteriorated on the tenth day after admission and he diedsix days later.

CASE 3 A man, aged 31, an enamel worker, had been ingood health until three years ago when he began to haverecurrent episodes of vertigo, which was not related toposture, but without tinnitus or impairment of hearing.The vertigo had apparently been less frequent in the pasteight months although no treatment or investigation wascarried out during this time. Three weeks before admissionthe patient had an acute attack of bilateral and verticalheadache, which was very persistent and dull and achingin character. He also complained of diplopia, especiallyon looking towards the left side. He was admitted to theQueen Mary Hospital on 6 December 1961.On examination, there was paralysis of the left lateral

rectus. Since the patient had been an enamel worker for 30years, chemical investigation of blood and radiographicalstudies of the epiphyses for lead absorption were donewith negative results. Radiographs of the chest and testsof the blood and cerebrospinal fluid for venereal diseasewere all negative. Pandy's test was positive: total protein30 mg./100 ml.; sugar 40 mg./100 ml.; chloride 680mg./100 ml. Bacteriological examination of the cerebro-spinal fluids taken on the fifth, seventh and 14th days afteradmission showed C. neoformans in smears and bycultural methods on each occasion. The first cerebro-spinal fluid specimen was also examined for tuberclebacilli by smears, culture, and guinea-pig inoculation,but with negative results.The patient was treated with amphotericin B, 1 mg. per

kg. daily, given intravenously. In spite of the treatment,which had been given for 10 days, he died on 3 January1962. Escherichia coli was isolated from blood three daysbefore death but C. neoformans was not found, showingthat the fungus had not been disseminated into the bloodstream. Permission for necropsy was not available.

Strain

BACTERIOLOGY

The cerebrospinal fluids of the three cases wereexamined in the same manner. The sediments of thecentrifuged fluids were first examined by directsmears mounted in Indian ink and in lactophenolcotton blue and afterwards in smears stained byGram's method. They were then cultured onSabouraud's glucose agar incubated in duplicate at37°C. and at room temperature. Cultures on freshand heated human blood agar media incubated at37°C. were also made in order to detect otherpathogenic bacteria. After isolation, subcultures inappropriate media were incubated at 37°C. to beexamined for mycelium formation, for sugar fer-mentation reactions, for nitrogen and carbonassimilations, for urea hydrolysis, for indole pro-duction, and for nitrate reduction. Finally, tests foranimal pathogenicity were carried out by inoculatingthe isolates into white mice and guinea-pigs. Theresults of these tests are summarized in Table I.

Thickly encapsulated, budding, yeast-like cellswere seen in direct smears of the sediments of thecentrifuged cerebrospinal fluids. The yeast-like cellswere round, rarely oval, and varied a great deal indiameter. Excluding the capsule, the diameter variedfrom 3 to 25,t but the majority were about l10t indiameter. The cells were Gram positive. The capsulescould be seen clearly in wet smears mounted in lacto-phenol cotton blue examined under subdued light(Fig. 1) but were also conveniently demonstrated byIndia-ink preparations (Fig. 2).The growth was very profuse on Sabouraud's

glucose medium incubated at 37°C. Well-developedcolonies about 2 to 5 mm. in diameter, which wereheaped up, dome-shaped, creamy white and mucoid(Fig. 3), formed within 48 hours. Smears from thesecultures showed encapsulated budding yeast-likecells not unlike those seen in direct smears, exceptthat the capsules were narrower and not so welldefined and that the cells were more regular in size.Upon further incubation, the colonies changed from

3LE IRESULTS OF BIOCHEMICAL AND PATHOGENICITY TESTS OF THREE STRAINS OF

C. NEOFORMANS

Growth Indole Reduction Urea KNO3 Carbon Assimilationfrom Pathogenicityat 37'C. Production of Nitrate Hydrolysis Assimilation for Mice

to Nitrite

X °t

I (culture from case 1)11 (culture from case 2)1II (culture from case 3)

± ± + + + ± -+ + + +- ± ± _ -± + + + + + -

+

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C. T. Huang, P. C. Wong, and C. H. Chan-Teoh

t

_.,: :$ b

Z,-.

tr

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0_

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FIG. 1. C. neoformans from cerebrospinal flumounted in lacto-phenol cotton blue. Capsulesdistinguishable. x 600.

creamy white to brown and were drier. Myceliumwas not formed even after prolonged incubationboth at 37°C. and at room temperature, nor was it

v formed in corn meal-agar subcultures. Though thegrowth was slower at room temperature between

r 25 and 28°C. it was just as good as at 37°C. uponfurther incubation. Subsequent tests in broth cul-tures with an initial pH of 7-4 showed that the pH ofthe medium increased with time, reaching 8-08-3after one week's incubation at 37°C. for cultures ofall three strains.Growth on fresh and heated blood agar media

yielded poor growth. Only minute pinpoint coloniesmeasuring less than 1 mm. in diameter developed intwo or three days' time. The colonies were notappreciably bigger even after many days of incuba-tion. No haemolysis was noticed from growth in

%t fresh blood agar and haemodigestion was not seenin heated blood agar medium.

Biochemically, none of the three strains producedindole from peptone broth, or reduced nitrate tonitrite. They produced acid but no gas in fluidmedium containing glucose and mannose in two days.No change was observed in maltose, mannite,sucrose, salicin, lactose, and galactose after 14 days'incubation at 37°C. For carbon assimilation and

h nitrogen assimilation tests, the methods of Lodderand Kreger van Rij (1952) were followed. In the latter

rid of case 2 potassium nitrate (Analar) was used. A negativeare clearly control was always included by inoculating the cul-

tures onto each batch of the basal medium. The

FIG. 2. C. neoformans from cerebrospinal fluid of case 2 FIG. 3. Culture of C. neoformans from case 2 in Sabou-in Indian-ink preparation. x 275. raud's glucose agar incubated at 37°C. for 48 hours.

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results showed that carbon was assimilated fromglucose, mannose, maltose, mannite, sucrose, andgalactose but not from lactose and salicin. Nitrogenwas not assimilated from potassium nitrate by anyof the three strains. All strains hydrolysed ureawithin 24 hours of incubation. The results of all thebichemical tests are set out in Table I.

Sensitivity tests in vitro against antifungal agentssuch as actidione, nystatin (Mycostatin), andamphotericin B (Fungizone) were carried out.Because amphotericin B was not available whenstrains 1 and Il were isolated, their stock cultureswere used for testing. The antifungal agents wereincorporated in melted Sabouraud's glucose agar(pH 7T0) at 45°C., poured on plates, and hardenedimmediately in the refrigerator. The concentrationsof each agent used were: actidione 24, 2-4, and 0-24,tg./ml.; nystatin 100, 10, and 1 unit/ml.; amphoteri-cin B 10, 1-0 Hg./ml. The inoculum was one loopfulof a 24-hour culture of the test strain in glucosebroth (pH 7-0) streaked all over the surface of theplate. Glucose agar plates without antibiotics wereseeded for control. All plates were incubated at 37°C.for two days, and results were read with the aid of adissection microscope. The results are presented inTable II. All the three strains were inhibited byamphotericin B within the range of inhibitionin vitro studied elsewhere. Strains 1 and II were moreresistant to actidione than strain III and all three wereunusually resistant to nystatin. According to theinformation provided by the manufacturer theactivity of nystatin decreases when a suspension isstored at 37°C. The incubation temperature in ourtest definitely affected the result. On the whole,strain III differed from the other two strains inresponse to these antifungal agents. It was moresensitive to actidione but was more resistant tonystatin and amphotericin B than the other twostrains.Benham (1956) has pointed out that the degree of

invasiveness for mice is an important diagnosticcharacteristic for species of Cryptococcus and thatonly C. neoformans is pathogenic. All our strainswere inoculated into white mice soon after isolation.For each isolate eight mice were inoculated, fourreceiving intraperitoneal and the other four intra-cerebral injections. Thesizeofthe inoculum permouse

for intraperitoneal injection was 0 5 ml. of a 24-hourglucose broth culture and that for intracerebralinjection 0-1 ml. of the same culture. The progress ofthe infection among the animals was very irregular,but on the whole those injected intraperitoneallydied within two weeks of inoculation and thoseinjected intracerebrally within 10 days. Necropsyfindings in the mice were essentially the same for allthree strains. The lungs were consolidated but theextent differed in each individual mouse. The brainswere soft and congested or normal in appearance. Afew of the animals showed a slightly enlarged spleen.The livers and kidneys were apparently normal ingross appearance. Those injected intraperitoneallyoften showed congested vessels of the abdominalwall and increase of peritoneal fluid, which wasgelatinous. Histological study of sections of theorgans, including the brains, showed numerouscryptococcal cells in the tissue spaces and the lumenof the vessels but the cellular reactions were mild incontrast to the overwhelming number of organismspresent.One locally bred and one Dunklin-Hartley strain

of guinea-pig, weighing about 350 to 400 g., wereinoculated with each of the strains of C. neoformansisolated. One millilitre of a 24-hour glucose brothculture was injected subcutaneously into the leftgroin of each animal. The Dunklin-Hartley guinea-pigs died within one month of inoculation, varyingfrom 25 to 30 days for strains I, II, and III res-pectively, whereas the locally bred animals showed aninconsistent result. That inoculated with strain I died40 days after inoculation, whilst those inoculatedwith strains II and III survived for more than twomonths. Post-mortem examination of the Dunklin-Hartley guinea-pigs thus infected showed largenumbers of cryptococcal cells in the lungs andspleens microscopically, less so in the kidneys. Therewas little tissue reaction in the form of proliferationof the reticulo-endothelial cells and the brains andlivers showed no change. Though the number ofguinea-pigs used for our test was small, it is clear thatvariation between strains in susceptibility of guinea-pigs to infection is an important factor.From the above findings the diagnosis of Crypto-

coccus neoformans of the three strains is justified, forall of them fulfilled the criteria laid down by Littman

TABLE IIEND-POINT OF INHIBITION IN VITRO OF C. NEOFORMANS TO ANTIFUNGAL AGENTS

Actidione (jug./ml.) Nystatin (units/ml.) Amphotericin B (,.g./ml.)

Strain IStrain IIStrain IIIEnd-point according to reference

24 102-4 100-24 1000-24 (Littman and Zimmerman, 1-5 (E. R. Squibb & Sons,1956) technical leaflet, 1961)

0 10 11*00 03 to 0-6 (Seabury and Dascomb,1960)

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and Zimmerman (1956). We had no opportunity toexamine serologically the patients' sera for capsularreacting antibody or agglutinating antibody. Alsowe had not tested the serological relationship of ourstrains by the above-mentioned reactions. However,guinea-pigs inoculated with living cultures of thethree strains were skin-tested for allergic reactionswith heat-killed cultures of homologous and heter-ologous strains. The test was carried out as followsOne Dunklin-Hartley guinea-pig was inoculated

subcutaneously with 05 ml. of a 24-hour glucosebroth culture of each of the three isolates. Threeweeks later the animal was inoculated intradermallyinto separate areas with heat-killed cultures (60°C.for one hour) of the three strains. Similarly heat-killed cultures of C. neoformans innocuous andCandida albicans were injected into other areas ascontrols. Skin reactions appeared after two days notonly on areas injected with the three strains of C.neoformans but also on those injected with C.neoformans innocuous and Candida albicans.

DISCUSSION

In all eight cases of cryptococcosis reported in China,there were symptoms of meningeal involvementbefore death and in seven of them C. neoformans hadbeen isolated from the cerebrospinal fluid (Cheng,1956). In our series, all three cases ended fatallyafter the development of meningeal symptoms. Itappears that cryptococcal meningitis or menin-goencephalitis is still the most frequent cause ofdeath in this fungal infection. The course of thedisease is variable.

C. neoformans is widely distributed in nature, forexample, in milk (Carter and Young, 1950), in soil(Emmons, 1951), in various animals (Littman andZimmerman, 1956), and in pigeon's nests (Kao andSchwarz, 1957). The organism is extremely resistantto dessication. According to Cheng (1956), drypowder of two strains of C. neoformans stored forone year and eight months was still alive upontransferring it to fresh cultural medium. He alsofound that dried sputum containing the organismgave growth upon inoculation to cultural medium ayear afterwards. The high resistance to drying cer-tainly has some significance in its epidemiology.Inhalation of dust from a contaminated source is themost probable mode of infection. However, therewas no definite evidence to link the occupations ofthe patients reported here to these sources of infec-tion. Emmons (see McCullough, Louria, Hilbish,Thomas, and Emmons, 1958) has pointed out thatcryptococcosis was a non-contagious mycosis; thatnone of the other members of the big family of case2 had the same complaint supports this point.

Clinically, cryptococcal meningitis and meningo-encephalitis are usually mistaken for tuberculousmeningitis. This is especially true in the Far East,where tuberculosis is still very common.The presence of thickly encapsulated spherical,

budding, yeast-like cells in the sediment of the centri-fuged cerebrospinal fluid is suggestive of crypto-coccosis. The capsule is best demonstrated by anIndian-ink preparation but a wet smear mounted inlacto-phenol cotton blue is also useful, because itdigests the red blood cells and leucocytes leaving thecryptococci and their capsules intact. The sphericalshape of the fungus is an important criterion, for ithelps in the histological and cultural diagnosis ofcryptococcus because most other yeast cells are egg-shaped or elliptical in outline. This point had beenemphasized by Emmons (see McCullough et al.,1958).

It is not difficult to culture C. neoformans fromthe cerebrospinal fluid, and Sabouraud's glucoseagar is the medium of choice. Duplicate sets incu-bated at 37°C. and at room temperature might be setup in case other fungi are looked for. As a rule, C.neoformans grows much faster at 37°C. than at roomtemperature on primary isolation. The differentiationof C. neoformans from the saprophytic cryptococcihas been fully discussed by Benham (1956). In viewof the irregularity of the results in the sugar fermen-tation test, the more reliable carbon assimilation testshould be used instead. Though the number ofstrains tested by us was small, our results were in fullagreement with those of Benham. All our strainshydrolysed urea in Stuart's medium within 24 hours.Seeliger (1956) found that this test was useful toscreen the genus Cryptococcus from the urease-negative Candida. It is, therefore, useful for primaryidentification of Cryptococcus isolated from mixedcultures. Nevertheless, animal inoculation is essentialto discriminate C. neoformans from the saprophyticCryptococcus species. The white mice, which areregularly susceptible to C. neoformans and succumbin a shorter period of time, are the animals of choice.

Actidione (Carton, 1952), nystatin (Brown andHazen, 1957), and amphotericin B (Louria, Feder,and Emmons, 1957; Biddle and Koenig, 1958;Fitzpatrick, Rubin, and Poser 1958; Seabury andDascomb, 1960) are all drugs which have beenemployed with some success in the treatment ofcryptococcosis. Sensitivity tests of our strains in vitroshowed that they were sensitive to these antifungalagents. In clinical practice, actidione and nystatin aredisappointing in the treatment of cryptococcalmeningitis, because they do not reach appreciablelevels in the cerebrospinal fluid and central nervoussystem after intramuscular or intravenous injection(Littman and Zimmerman, 1956). In addition,

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nystatin is fairly toxic when administered parentally.Amphotericin B appears to provide promising resultsif it is used early. Treatment of cryptococcosis withamphotericin B has been observed to be successfulby many workers. Only case 3 in our series wastreated with amphotericin B, but no clinical improve-ment was achieved owing to the advanced stage ofthe disease. The failure of amphotericin treatmentin systematic mycosis has been discussed fully by Utzand Andriole (1960).

We are grateful to Drs. H. L. Wen, Stephen Chang, andJ. Hunter for the clinical notes of the patients whosecases are reported in this communication. We thank theU.S. Summit Corporation (Panama), local agent of E.R.Squibb and Sons, N.Y. for supplies of nystatin (Mycos-tatin) and amphotericin B (Fungizone); Dr. M. Silva,Department of Dermatology, College of Physiciansand Surgeons, Columbia University, New York, U.S.A.,for subcultures of C. neoformans innocuous.

REFERENCES

Benham, R. W. (1956). Bact. Rev., 20, 189.Biddle, A., and Koenig, H. (1958). Arch. intern. Med., 102. 801.Brown, R., and Hazen, E. L. (1957). Trans. N. Y. Acad. Sci., Ser. 2,

19, 447.Carter, H. S., and Young, J. L. (1950). J. Path. Bact., 62, 271.Carton, C. A. (1952). Ann. intern. Med., 37, 123.Cheng, W. F. (1956). Chin. med. J., 74, 374.Fitzpatrick, M. J., Rubin, H., and Poser, C. M. (1958). Ann. intern.

Med. 49, 249.Emmons, C. W. (1951). J. Bact., 62, 685.Kao, C. J., and Schwarz, J. (1957). Amer. J. clin. Path., 27, 652.Littman, M. L., and Zimmerman, L. E. (1956). Cryptococcosis.

Grune and Stratton, New York and London.Lodder. J., and Kreger van Rij, N. J. W. (1952). The Yeasts a

Taxonomic Study. Interscience Publishers, New York.Loh Siew Whye (1951). Proc. Alumni Ass. King Edw. VII Med. Coll.,

4, 143.Louria, D. B., Feder, N., and Emmons, C. W. (1956-57). Antibiotics

Ann. pp. 870-877.McCullough, N. B., Louria, D. B., Hilbish, T. F., Thomas, L. B., and

Emmons, C. (1958). Ann. intern. Med. 49, 642.Pallis, C. (1949). Proc. Alumni Ass. King Edw. VII Med. Coll., 2, 452.Russell, R. W. R., and Dean, D. (1957). Brit. med. J., 2, 627.Seabury, J. H., and Dascomb, H. E. (1960). Ann. N.Y. Acad. Sci.,

89, 202.Seeliger, H. P. R. (1956). J. Bact., 72, 127.Utz, J. P., and Andriole, V. T. (1960). Ann. N. Y. Acad. Sci., 89,277.

The July 1963 IssueTHE JULY 1963 ISSUE CONTAINS THE FOLLOWING PAPERS

Inherited enzyme defects: a review T. HARGREAVES

A cytological and histological study of acute premyelo-cytic leukaemia JEAN BERNARD, J. LASNERET, J. CHOME,J. P. LEVY, and M. BOIRON

Cellular types in acute leukaemia: diagnosis and signi-ficance F. W. GUNZ and A. F. BURRY

Agglutination reactions of human leucocytes JEANETTESCHULZ and HELGA MULLER

Fatal hepatic necrosis in glandular fever U. R. ALLENand B. H. BASS

A study of the vesical ganglia in children and the relation-ship to the megaureter megacystis syndrome and Hirsch-sprung's disease SIDNEY LEIBOWITZ and MARTIN BODIAN

Four cases of Waldenstrom's macroglobulinaemiaD'A KOK, D. N. WHITMORE, and R. W. AINSWORTH

Synergism between sulphonamide drugs and antibioticsof the polymyxin group against Proteus sp. in vitroF. E. RUSSELL

A new Pasteurella species (?) from a wound infectionJ. BRODIE and A. HENDERSON

A comparison of the measurement of gamma globulin incerebrospinal fluid by electrophoretic and immunologicalmethods JACK COLOVER, J. G. FEINBERG, ANNE TEMPLE,and MARIAN TOOLEY

Electrophoretic patterns of tumour tissue proteinsEMIDIO AFONSO

A rapid micro method for recording red cell osmoticfragility by continuous decrease of salt concentrationD. DANON

Technical methodsA new procedure for the identification of Pneumocystiscarinii cysts in tissue sections and smears A. M.CHALVARDJIAN and L. A. GRAWE

Separation of myoglobin and haemoglobin on a columnof dextran gel M. C. BERMAN and J. E. KENCH

A method for applying haemolysates on starch blocksfor electrophoresis P. BARKHAN and M. E. STEVENSON

A routine method for staining eosinophils in sputumRICHARD W. PAYNE and R. H. HILL

The Association of Clinical Pathologists: 70th generalmeetingInternational enzyme units and isoenzyme nomenclature

Book reviews

Copies are still available and may be obtained from the PUBLISHING MANAGER,BRMSH MEDICAL ASSOCIATION, TAVISTOCK SQUARE, W.C.I, price 18s. 6D.

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