A histrical review on the taxonomy of Cryptococcus neoformans was presented. And a total of 27 strains of C. neoformans, of which 26 were isolated from clinicl material and 1 from dropping of pigeon in Japan, were studied. After three to four weeks incubation on yeast extract-sucrose-biotin agar, 21 out of 27 strains did not produce a hypha at all, but showed only globular cells; 3 developed a germtube; two put forth short hyphae. The remaining 1 strain generated hyphae having clamp-connections, basidia and basidiospores; this strain was thus found to be self-fertile, while 26 others were all self-sterile. In the mating study of these strains, 24 were compatible and 2 were imcompatible. All of the strains “compatible” were α-type of Filobasidiella neoformans. None of a-type of F. neoformans and F. bacillispora were seen. By serotype, 26 strains were A-type and only one was the type A-D. The types B and C were nil. The type A-D strain isolated from dropping of pigeon in kyoto was self-fertile. In the United States, three “self-ferile” strains have so far been reported, however, all of them are those isolated from clinical lesions. This presents an impressive contrast to our strain isolated from natural substrate. Additional tests such as assimilation test, fermentation thest, urease test, DNase test, test on production of starch-like compounds or test on growth at 37°C were carried out. The results of all of these tests were consistent with standard strains.

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Public nuisance brought about by semi-wild pigeons domiciliating in common environment and public facilities not only is a menace to the human life environment economically as a social problem but also is of no little gravity in public health, as viewed from the ecological standpoint. Pigeons are generally regarded as peace envoys but there have been many reports purporting to demonstrate unimportant role played by flocks of pigeons in the epidemilogy of cryptococcosis with high mortality. Since a report of Emmons, rather constant, frequent occurrence of C. neoformans in pigeon dung has been demonstrated and confirmed by studies in many other countries as well as in Japan. While isolation of the organism from other sources such as other species of birds, mice, domestic animals, fruit juice, caviare and sea water of Osaka Bay has also been reported, it is generally recognized to be unquestioned that bird feces serve as natural habitat for the organism as its exogenous source of transmission. Recently, we have conducted a study to investigate the distribution of C. neoformans in nature and to explore and characterize by physical and chemical means the behavior of this fungus in pigeons with most profound relation to it, captured and maintained in the laboratory colonies.

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Antigenic analyses of four serotypes of Cryptococcus neoformans were carried out by absorption with reciprocal absorption experiments, and the antigenic patterns of four serotypes were established. The serotypes A, D, B and C demonstrated antigenic factors 1, 2, 3, 7; 1, 2, 3, 8; 1, 2, 4, 5; and 1, 4, 6; respectively. Comparative experiments by our system for serotyping of C. neoformans isolated from Japan indicated that 49 out of 52 strains were identified as serotype A. Remaining 3 strains exhibited antigenic factors 1, 2, 3, 7 and 8. Therefore, we designated them as serotype A-D tentatively. It was suggested that 8 kinds of factor sera were useful for identifying the serotype of C. neoformans isolates, and the majority of isolates in Japan were serotype A.

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Cryptococcosis can be classified into primary and secondary cryptococcosis. In Japan, the latter disorder is found much more often than the former. Primary cryptococcosis is usually observed during surgery rather than antopsy, but such occasions are relativiely few. Bearing these facts in mind, the authors studied the statistical characteristics of cryptococcosis from the pathological standpoint. Furthermore, the pattern of response from human subjects was also studied for both cryptococcal granuloma of the lung, including primary disorder, and secondary cryptococcosis.
1) Statistical Observation of Cryptococcosis: Some pathologists have reported their findings on autopsy statistics of deep mycosis. In this study, the autors made a statistical observation of cryptococcosis and other mycoses on the basis of the annual report of pathologic autopsy cases edited by the Japanese Pathological Society from 1958 to 1977. Of some 390, 000 autopsy cases reported in the report, mycosis was found in 1.67% Cryptococcosis, seen the third most often following Candidiasis and Aspergillosis, was found in approximately 10% of all mycosis cases.
2) Pathological Study of Cryptococcal Granuloma of the Lung: Cryptococcal granuloma is often found in the lungs, and its clinical diagnosis is fairly difficult. Patient with this disorder frequently undergo excision of the lung for lung cancer and are later diagnosed histologically as cases of cryptococcosis with granuloma. The pathology of this disorder is most often the subpleural formation of granulomata. In the textbooks of foreign countries, the granuloma in this disorder does not often develop central necrosis and form cavities. In Japan, however, it does not appear infrequent that central necrosis and cavity formation following the appearance of granulomata. Thus, the autors studied the nine cases of cryptococcal granuloma of the lungs reported in Japan to ascertain the mechanism of the granuloma and cavity formation. It is thought that the cryptococcal ganuloma in the lungs begins with an acinar change, that the completed granuloma has a simple pattern of reticulin fiber production, unlike the granuloma found in sarcoidosis, tuberculosis or other disorders and that the granuloma in this disorder is surrounded by a thick fibrous capsule. The authors assumed that the central necrosis and the cavity formation are caused by the vascular changes within the granuloma, in pariticular, circulatory disturbances by fungal emboli.
3) On Secondary Cryptococcosis: Hematopoietic diseases are often found as underlying diseases. In secondary cryptococcosis, the lungs, the central nervous system and the kidneys are involved in this disorder. Tissue reaction to this organisms is weak and as a result, the disorder is observed as a cystic lesion.

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We have been investigated human and experimental cryptococcosis histopathologically, and some specific and interesting findings were obtained concerning on morphology and staining behavior of cryptococcus cells in infected lesions, and an outline of the results are reported. Twelve necropsy cases and experimental murine cryptococcosis were submitted for study. The size of cryptococcus cells in cerebral and pulmonary lesions of human cases were measured with a micrometer. In the infected foci with relatively short clinical duration most of the cryptococcus cells were rather uniform in size and in shape, however in chronic granulomatous lesions remarkable difference in size and the frequent occurrence of aberrant forms of cryptococcus cells were observed. The size of the cryptococcus cells in the experimental infections was also variable with chronic course. These findings could be assumed as one of the reliable clues for the determination of the stage of cryptococcus infection. Infected tissue sections were stained with 1% aquous solution a Brankophore-P, and examined through fluoresecent microscopy. Cryptococcus cells in fresh lesions emit an intensive fluorescence, but the fluorescence of the fungus cells in chronic lesions was weak. In addition, relationship between the florescence intensity and the viability of the cryptococcus cells were tested in vitro. it was confirmed that the intensity of fluorescence of fungus cells was closedly correlated with the viability of the fungi.

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The fluorescent antibody (FA) technique has been already established as one of diagnostic and research tools in medical mycology. It is emphasized that the FA technique can be used for the rapid detection and identification of fungi in cultures, clinical materials and even in paraffin sections of formalin-fixed tissues. In addition to these applications, the indirect FA procedure is also valuable to detect and measure antibodies to fungi in sera and in other body fluids. In most instances, the FA reagents to fungi cross-stain other members of the genus and some of other heterologous fungi. However, extrageneric cross-reactivity, if present, can be removed by dilution or adsorption In its current state of development, the FA technique is a highly useful procedure for the diagnosis of cryptococcosis. Unadsorbed Crytococcus neoformans antiserum cross-reacts with other Cryptococcus species and Candida species. The cross-reactivity with Candida species is removed, by dilution of the FA reagents to C. neoformans or adsorption of the reagents by using C. albicans. In addition, the indirect FA technique is frequently used for the detection of cryptococcal antibodies in sera.

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Crytococcosis is a subacute or chronic pulmonary, systemic or meningeal mycosis by Cryptococcus neoformans. Involvement of the central nervous system with subacute or chronic meningitis is the most familiar form. First, in this symposium, 9 cases of cryptococcal meningitis at Juntendo University Hospital were reviewed, particular attention being given to symptoms and cerebrospinal fluid and culture findings and relation of these findings to the outcome of treatment. Second, cryptococcal meningitis in Japan, were reviewed for about seventeens years from Sept. 1961 to Dec. 1978. In this period, the total number of 225 cases were obtained with cryptococcal meningitis. The yearly incidence were ranging from 10 to 15 cases except 1962, 1965 and 1972. The male-to-female ratio, including five cases of whom no mention of sex and age, is about three to two, and the age pattern shows highest in the incidence in the 20's The mortality rate of patients with cryptococcal meningitis has decreased due to the use of amphotericin B and flucytosine, but this disease remains one of high mortality in Japan. The cure rate is low for the period from 1961 to 1972 (32.8%), but is higher after 1973 (45.8%) that may be attributed to the fact by the treatment with amphotericin B and flucytosine, and the concern of the medical field regarding this disease had increased. Concerning the clinical symptoms or cerebrospinal fluid were not particular. Coexiting disease occured in 113 of the 225 cases, malignant lymphoma in 21 (18.6%), SLE in 18 (16%), acute lymphocytic leukemia in 9, liver cirrhosis in 6, diabetes mellitus, acute myeloic leukemia in 5 and others.

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Forty two cases of cutaneous cryptococcosis were reported in Japan from 1958 to 1980, in which 20 cases were male and 22 female. Twenty six cases (61.9%) occurred between 20-40 years of age. There were 12 cases in which primary cutaneous cryptococcosis was localized and 7 cases spread systematically. Twenty one cases showed cutaneous manifestation secondarily and there was no detailed decription in 2 cases. Regarding the description of the lesion, acneform eruption manifested in 16 cases (34.0%) and nodular tumor in 14 cases. in the histopathologic picture, the Moore's 1st type showed good prognosis and 2nd type high mortality. The cutaneous lesions, when they were primary and localized were all 1st type and all cured. The survival rate is increasing in the 2nd type. This may indicate the early diagnosis and early treatment.

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Cryptococcus neoformans is a kind of common and pathogenetic fugi to involve the central nervous system. This disease is classified to three types from the clinical features: meningitis, meningoencephalitis and granuloma, and showed various symptoms. The diagnosis was sometimes difficult. In our 3 of 5 cases, inflammatory symptoms were not observed and disorientation, memory loss, diplopia, convulsion, pyramidal and extrapyramidal signs appeared. In 1 of 3 cases, symptoms of normal presure hydrocephalus were observed. In the other one case, non-communicating hydrocephalus was showed in CT scan. These patients were at first misdiagnosed as brain tumor or brain infarction in other hospitals. Generally, cryptococcosis of central nervous system is sometimes liable to diagnose as brain tumor or tuberclous meningitis initial stage. Bibliograpically, 17 cases were diagnosed to brain tumor and 4 of these cases were done craniotomy. For the diagnosis of cryptococcosis, Cryptococcus neoformans must be discovered in CSF, but the detection is not easy from the spinal puncture. There was a report that fungus was not detected by the repeated spinal puncture of 19 times. The ventricular puncture is the most available technique for the diagnosis of cryptococcosis, that is, our 3 cases were difinitely diagnosed by this technique rather than spinal puncture. When CSF by spinal puncture reveals the inflammatry findings without detection of the organisms, the ventricular puncture should be taken to detect it.

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So far immunodeficiency has been concerned in the cryptococcal infections, there were many cases of secondary infection occured in the case of the advanced stage of hematologic malignancies, paticularly in Hodgkin's disease. However, the exact etilogic aspects on the cryptococcal infection occuring in the immunodeficiency has not been clarified as yet. Few experimental results explained on the enhancing effects of cryptococcal infections in the deficiencies of cell-mediated immunity. Our experiments on cryptococcal infections in the mice, the immunosuppressive agents promoted death rate. Therefore the enhancing activity of cryptococcal infections in the immunodeficient host may be depended upon not only depressed of cell-mediated immunity but also compromised states introduced by many factors in the host.

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In the cases of cryptococcosis that we observed in autopsy in 1972, organisms were mostly present in the intrainsular or periinsular areas with necosis of the Langerhans islets. It was thought that these insular lesions produced a decrease of insulin secretion and secondary diabetes mellitus.
1. Clinical cases:
Case 1, 49-yr, M., a primary diabetic complicated with cryptococcosis showed numerous organisms in many islets and also degenerative changes of the islets. The diameter of the orgainsms in the islets were larger than that of the extrainsular. In case 2, 65-yr, F., a high fever was of chief complain. Her glucose tolerance was impaired and insulin secretion to glucose was decreased in the coures of her illness. These results might be induced by organisms in the islets and liver. In case 3, 76-yr, M., organisms were histologically seen in the islets. The diameter of the organisms was same as that in extrainsular areas.
2. Experimental study:
Fifteen days after the injection of organisms into the tail of rats, glucose tolerance were examined by means of loading 0.5g/kg glucose intravenously. In one out of three animals, organisms were seen in the intrainsular and periinsular areas in three of ten islets, while very few in the extrainsular areas. In the rats of experimental cryptococcosis, secondary diabetes was recognized with a remarkable decrease in glucose tolerance and in insulin secretion. This result suggested the similar occurrence of the secondary diabetes in the patients with cryptococcosis.

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The statistical survey was performed on the inpatients with cryptococcal infection at Kyushu University Hospital during the period from 1975 to 1979. In the studied period 18 cases of cryptococcosis were recorded, and 2 to 6 cases per annum were detected. In the whole group 11 men and 7 women were affected, and their mean age was 50 (range 15-84). Forteen cases of them were considered as a possible hospital-acquired cryptococcosis. The main predisposing conditions were malignant lymphoma and leukemia. The mortality in the possible hospital-acquired cryptococcosis was 86 per cent (12/14 cases) with a high rate of relapse. The figures reported here emphasize that cryptococcosis should be regarded as one of the main opportunistic fungal infections.

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Cases with cryptococcosis investigated were divided into two groups according to the periods experienced. Group A (21 cases) was from 1952 to 1966 and group B (18 cases) was from 1969 to 1979. These cases were obtained from our hospital and some from various medical institutes in Chugoku-Shikoku districts. In group A, 13 cases were primary cryptococcosis and 8 were secondary. Premortem diagnosis was done in 13 cases, and 8 were found after autopsy. Only 4 cases with primary cryptococcosis were successfully treated with amphotericin B, the doses of which ranged between 418mg and 2, 094mg. In group B, 6 cases were primary cryptococcosis and 12 were secondary. Premortem diagnosis were made in 12 cases, and the other 6 cases were confirmed after autopsy. Seven cases including 2 secondary cryptococcosis were so far successfully treated. Chemotherapy included amphotericin B in 5 cases, amphotericin B with 5-FC in 3 cases, and only one case was treated by 5-FC alone. The total amounts of amphotericin B in survived cases were between 400mg and 1.823mg. Three cases with pulmonary cryptococcosis and one with cutaneous cryptococcosis were surgically removed followed by amphotericin B administration. The prognosis of cryptococcosis is generally unfavorable, especially in secondary cases. However, in cases with later period of group B, the prognosis was better, even in secondary cryptococcosis, than that of group A. The key points for the successful treatment of cryptococcosis are early diagnosis and early treatment in consideration with the way of administration of the enough amount of amphotericin B by avoiding the side effects including fever, hepatorenal dysfunctions and so on.

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A focal primary pulmonary lesion of cryptococcosis is an indication of surgical incision. Fifty-three cases of pulmonary cryptococcosis, including our series of 3 cases, were reported in Japan from 1964 to 1980. The review of those cases revealed that successful surgical resection was performed in 40 patients, 24 cases of lobectomy, 8 cases of segmentectomy, 5 cases of partial resection and 3 cases of lung biopsy. A half of them was diagnosed preoperatively as lung cancer. Eleven patients were treated solely with antifungal chemotherapy and 2 patients were free of any treatment. Among 40 patients operated, 3 cases were treated with antifungal chemotherapy prior to the operation and postoperative treatment with antifungal chemotherapy was performed in 16 cases. As the transbronchial lung biopsy has been increasing the usefulness, 23 cases were diagnosed as pulmonary cryptococcosis prior to the initiation of treatment. Nine patients were operated among these patients, 6 cases with discrete circumscribed masses without the sign of meningeal involvement, 1 case which fails to respond to antifungal chemotherapy showing persistent positive culture, 1 case of local recurrence and 1 case without noticeable alterations of clinical manifestation.

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The histopatological examination was performed in search of the incidence, distribution of organ involvement and gravity of deep fungus infections in 82 cases (54 cases of acute leukemia, 8 cases of chronic leukemia and 20 cases of lymphoreticular disease), which were autopsied at our department during the 6 years from 1974 to 1979. The deep fungus infections were noted in 36 (66.7%) out of all cases with acute leukemia. Fifteen cases of them had two kinds of fungus infections. Among those in acute leukemia, candidiasis was the most frequent fungus infection being revealed in 26 out of 36 cases (72.2%), and was followed by aspergillosis (18 cases), mucormyosis (6 cases) and cryptococcosis (1 case). However, in candidiasis most cases did not show serious fungus infections, while 5 cases represented disseminated type and only one case showed severe lung involvement. On the other hand, mucormycosis unexceptionally represented large pulmonary hemorrhagic infarction and was fatal to all the 6 cases. Also, aspergillosis commonly involved the lung and was fatal to the two thirds of the 18 cases, Fungus infections were also demonstrated in 3 cases (37.5%) with chronic leukemia and 8 cases (40%) with lymphoreticular disease, although the number of the autopsy cases was small.
Finally, an evaluation of the clinical laboratory data was performed, and it was revealed that severe lymphocytopenia (less than 500/mm³) predisposed greatly to fungus infections. Therefore, it is assumed that deep fungus infections must be firstly suspected when marked lymphocytopenia is observed in the treatment of patients with leukemia and lymphoreticular disease.

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Ketoconazole (KCZ), a new water soluble oral imidazole, showed a broad antifungal spectrum in vitro including various dermatophytes, dimorphic fungi and pathogenic yeasts. Candida albicans, however, proved to be relatively insensitive to this drug and minimal inhibitory concentration (MIC) was determined as 50μg/ml for this fungus. With mice, 14μg/ml of the peak blood level was reached after a single oral administration of 80mg/kg of KCZ. Contrary to these observations, high survival rates were recorded in the experimental chemotherapy of murine candidiasis even with a single, relatively small dose (less than 50mg/kg) of the drug by intraperitoneal as well as oral administration. Detailed studies on the anti-Candida activity of KCZ in vitro revealed that the drug caused some inhibition of C. albicans growth at the concentration far below the minimal inhibitory concentration. Very low concentrations of the drug significantly delayed germ tube formation and subsequent development of pseudomycelia of C. albicans. In consequence, mouse peritoneal exudate cells were able to phagocytose readily C. albicans cells in the presence of KCZ as determined by ³H-uridine uptake. Furthermore, the intracellular killing rate was higher with KCZ treated C. albicans than that with untreated organisms. The conclusion was thus drawn that the excellent in vivo effectiveness of KCZ to C. albicans infection, despite of its relatively low in vitro activity, might be at least in part attributed to the inhibition of germ tube formation at low concentration and to the enhancement of phagocytotic and subsequent intracellular fungicidal activity of macrophages.

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The conidiogenesis of Geotrichum candidum grown on Czapek agar containing 1% yeast extract was examined by electron microscopy. The results showed that the entire wall of the fertile hypha is incorporated into the conidial wall and also that increase in thickeness of the inner wall layer and differentiation of crosswalls are likely to be due to new wall synthesis which occur during conidium formation. Furthermore, the chemical composition was analysed for the purified conidial walls, and was compared with that of haloarthric conidial walls produced by Trichophyton mentagrophytes.

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