Cryptococcosis is a systemic mycosis that continues causing a high number of infections in AIDS patients, mainly in not industrialized countries. The disease produces a great concern not only because of the clinical manifestations but also because of the high mortality associated with it. In 2009, Park estimated a million new cases yearly, mostly in sub-Saharan Africa (more than 700,000) with a mortality of about 600,000.62,71 Cryptococcosis in Latin-American countries account for a great number of cases, but there are only estimations of the prevalence of this mycosis based on information from research groups and not from national statistical data. In a recent epidemiological surveillance in Colombia, which covered the 1997–2014 period, a total of 1837 cases from different regions were reported, 76.9% (1413) in HIV-positive-individuals.30 Cryptococcosis is not a notifiable disease in our country and no reliable data about the real incidence of this pathology in Argentina is available. An Argentine publication accounted for a total of 105 cases widespread in the country in 1981–1990.6 A recent research done by the “Laboratorio Central de Redes y Programas” of Corrientes province informed about 26 detected cases between 2008 and 2013 in four general hospitals and one pediatric hospital. On the outskirts of Buenos Aires, 106 HIV-positive-patients (128 episodes) were attended between 1996 and 2007 at the Paroissien Hospital.53

Cryptococcosis is the most prevalent systemic mycosis in HIV+positive patients in the F.J. Muñiz Hospital. In the last years an average of 60 new cases have been diagnosed yearly, similar to those corresponding to P. jirovecii pneumonia and almost two fold the disseminated histoplasmosis. Almost half of the cryptococcosis diagnosis in Buenos Aires city and 15% of those cases from the whole country are made in this Infectious Diseases Hospital. These data show that this casuistic is very high compared to those of other institutions in Argentina and Colombia. The main risk factor in this cohort was the HIV infection (98%); this percentage was lower (76.9%) among the patients of a Colombian study in an 18 year-period.30 The high percentage of HIV-positive-patients could be explained by the fact that the F.J. Muñiz Hospital is an institution devoted to infectious diseases that assists most of the aids individuals requiring hospitalization in Buenos Aires city; on the contrary, patients with other type of immunodeficiency are habitually attended in other institutions.

Male/female rate of the presented cases was 3:1, quite similar to the findings in Paroissien Hospital (70.1% men), lower than the rate obtained in Colombia on 526 cases (83.5% males), lower than in the surveillance carried out in 1997–2014 (where rates varied from 4.7:1 to 3.1:1), and higher than the cases diagnosed in Corrientes province (2.6:1) among HIV+positive patients.15,53 The average age of the cohort studied increased through the 6 analyzed quinquennia as it happened with the HIV+positive population attending the Hospital. The mean age in the whole period was 34.1 years, with 26.7 years in the first quinquennium and reaching 38.5 years of age in the last one. In the Colombian population, 74.9% of the cases were between 21 and 50 years, and in Corrientes the median age was 38.5, the same as ours in the last quinquennium. In the cohort of Paroissien Hospital the median age was 34 years old.15,30,31,53 No statistical difference in age was found between both sexes (p=0.45). Most of the patients were in the late stages of HIV infection with a CD4+ lymphocyte median count of 35 cells/μl (interquartile range 15–70 cells/μl), being a population at high risk of suffering cryptococcosis. As it was expected, CSF was the sample rendering positive results in first place, followed by blood cultures, in consistence with previous findings in other investigations.6,46,53 The increase in the proportion of positive blood cultures and decrease in CSF positive cultures seen during the 6th quinquenium is probably due to an earlier diagnosis. Direct culture of all the samples (except blood and bone marrow aspiration) onto sunflower agar favored and accelerated the identification of this microorganism (presence of melanin pigment) and allowed the differentiation from other yeasts unable to produce melanin in materials like respiratory secretions, urine, biopsies, etc. Moreover, the quick urease detection method on filter paper brought about fast information and contributed to the early diagnosis of this mycosis.8,48

The Mycology Unit does not have proteomic equipment (MALDI-TOF) for species identification, thus yeasts belonging to the species C. neoformans and C. gattii were phenotypically differentiated by conventional methods using GCB and Salkin medium.43,64 All the isolates from HIV+positive patients were C. neoformans except for two C. gattii (0.1%). The last species was the causing agent in four HIV-negative patients (9.7%). Recently, the molecular identification by PCR-RFLP of URA5 gene in isolates from several periods included in this study was carried out. In this cohort 91.7% of C. neoformans isolates belonged to VNI genotype as it was found in other investigations. In 2003, the research performed with Spanish and Latin American environmental and clinical isolates, showed that 29/33 (87.9%) samples from HIV-positive patients from the F.J. Muñiz Hospital were VNI, 2 VNII and the remaining 2 VNIII, Considering all the Argentinean isolates, 57 (82.5%) were VNI, 8.9% VNII, and 3.6% VNIII.51 Another investigation on C. neoformans isolates in Corrientes province found that 15/18 cases belonged to VNI genotype, 1 to VNII, and 2 to VNII-VNIV hybrid.15 In the Perrando Hospital (Chaco province) 15/16 isolates from HIV-positive patients were VNI and 1 C. gattii VGI.16 A global research carried out with clinical and environmental isolates in different world regions showed similar results: VNI genotype was the most prevalent all over the world and 60/87 isolates from Argentina were VNI, as 71% of Latin-American isolates.21,33. On the other hand, a recent publication from Seville, Spain, showed that 64% of 28 isolates from 12 HIV-positive patients were C. deneoformans or C. deneoformans × C. neoformans hybrids.35

Another useful tool in the diagnose of the disease and the progress of the patients was the detection of CrAg in serum and CSF. In this study LA method was always used with the same kit (IMMY), with diagnostic and evolution control purposes. In order to compare the results it is very important using the same equipment since it has been shown that there is a marked difference in sensitivity among different commercial kits, which makes the comparison of data impossible.29,39,67 Lateral flow chromatography (LFA) kit (IMMY) began to be commercialized in Argentina three to four years ago. There are many publications that demonstrate its great sensitivity (superior to LA) and specificity.4,41,42,45,66,73 Currently, LFA is used in the Mycology Unit as a diagnostic tool in HIV-positive patients at risk of suffering cryptococcosis. If this test brings a positive result, the titer is determined by LA and different samples (especially CSF and blood) are further studied to microbiologically confirm the mycosis. Twenty years ago a research was carried out in order to make an early diagnosis of cryptococcosis in patients at risk (CD4+ counts <300 cells/μl, fever, without meningeal symptoms). It was done using LA and EIA tests on 193 patients. Seroprevalence of CrAg was 6.7% (13/193 patients) and only in three of them the cryptococcosis was microbiologically confirmed.58 CrAg titer in serum and CSF is useful as an indirect measure of fungal load; during the follow up period the titer decrease is consistent with the patient improvement. In our study, this fact was more evident with CSF samples because low titers corresponded to negative cultures (67.4% of negative CSF showed titers ≤1:10 in the second month after diagnosis) even when Indian ink still showed encapsulated yeasts. Serum titers diminished more slowly and more time was required to obtain negative results.3,13,29,36 This long follow up of CrAg titer in serum and CSF allowed us to observe negative results of these tests in patients after 18 or more months of secondary prophylaxis and HAART.59

The clinical symptoms and the location of the lesions were similar to the data presented in other publications.34,47,54,55,64 Meningoencephalitis was the most frequent clinical form (90% of patients), and measuring opening pressure at diagnosis is a key parameter to assess the progress. Among the patients in whom this information was available, 78.9% had a high intracranial pressure, which is a sign of poor prognosis if not controlled adequately in the first days of hospitalization, as demonstrated in previous research.11,24,27 Cerebral CT scan without contrast rarely shows pathologic findings; nevertheless brain magnetic resonance without gadolinium seems to be more sensitive and will show CNS images compatible with meningeal cryptococcosis. In a reduce number of cases they are observed as images with high signal intensities (at T2 and FLAIR), and usually correspond to enlargement of Virchow-Robin spaces, which are occupied by mucoid-protein material, affecting basal ganglia, perivascular parenchyma and, occasionally, the protuberance. Just exceptionally solid occupying lesions can be observed.26

Lung lesions appeared in 40% of the patients, but a great number of them also suffered other respiratory diseases (tuberculosis, atypical mycobacterial infection, P. jirovecii pneumonia, bacterial pneumonitis). The high frequency of respiratory involvement is due to the severe immunological compromise of this group of patients (the majority of them presented CD4+ cell counts ≤50/μl).40Cryptococcus was isolated from 82/108 respiratory samples as not always those samples were referred to a mycological study. The sunflower medium for the primary isolation of Cryptococcus in this kind of samples is notably useful since it allows brown colonies to be seen even when other yeasts are present in the same sample.48,61

Like in most Latin-American countries, 5FC has not been available for more than 2 decades in Argentina so induction therapy consisted of AMB alone. From 2010 onwards, induction treatment has been the association of AMB (0.7mg/kg daily, I.V.) with oral FCZ (800mg/day), as recommended by IDSA guides. This antifungal combination was more effective than the use of AMB alone, and cultures became negative in 3–4 weeks, as it was found in a previous research. Consolidation therapy continued with FCZ (800mg/day) and the results were very promising.49 Another investigation associated AMB with a lower dose of FCZ (400mg) during 2 weeks; the patients were followed up for only two months, and the results were not successful enough.12,69 In a meta-analysis performed by Campbell et al. the authors pointed out that in 35 investigations no evidence of a decrease in the mortality rate was observed when AMB was administered together with 5FC; moreover, they did not find any additional benefit in the AMB+FCZ combination.14 On the other hand, studies considering time and culture negativization rate found that AMB+5FC is a more effective treatment.63 In an attempt to elucidate if glucocorticosteroids could reduce mortality as in other types of meningitis, dexamethasone was combined with AMB+FCZ. The results were discouraging as there were more side effects, higher mortality rates and slower CSF clearance when compared with the control group.7 The change from induction therapy to consolidation therapy was based on CSF culture results (conversion from positive to negative). Interestingly, the cryptococcal antigen concentration in CSF was coherent with the result in the CSF culture, and in many occasions Indian ink showed encapsulated yeasts despite the low titer CrAg in CSF, thus cultures resulted negative. In this study the count of colony forming units (CFU) of CSF cultures were not carried out as a parameter to monitor fungal load and early fungicidal activity like in other investigations.63

High intracranial pressure should be controlled, especially within the 5 first days. This parameter has been taken into account considering it was previously demonstrated that when pressure continues above normal values during the mentioned period, mortality risk increases (OR 7.23, 95% CI 2.53–20.14).26 Serial lumbar punctures, even more than one a day, were useful to normalize the opening pressure in many patients.28 In those cases in which this method was unable to control the opening pressure, a ventricle-peritoneal shunt was performed with good clinical results.24

In this study, the decrease in the mortality rate (especially evident in the last quinquennium) was probably due to the control over the intracranial pressure, the combined therapy and the early diagnosis. On the other hand, the number of resistant isolates (MIC above ECV) was scarce and the MIC values obtained were similar to those found in VNI strains from Brazil.68 Therapeutic response and outcome were independent from MIC values, as it was seen in Colombian patients1 but different from findings that showed a correlation between the treatment failure and MICs≥16μg/ml in Spain.2 Nevertheless, heteroresistance should be considered in those patients with delayed negative CSF cultures.18

In this cohort the frequency of cryptococcosis in HIV-negative patients was very low (2%). In countries at other latitudes, like China or Iran, this condition is more frequent in HIV-negative individuals, probably due to genetic factors related to immunity (Han population in China). C. neoformans VNI and C. gattii VGI genotypes are prevalent there, in the manner of Australian isolates.33 Most cases corresponded to renal transplanted patients, and according to some publications it is important to consider the possibility of cryptococcosis in of organ donor transmission. Transplant candidates with cirrhosis should also be considered at risk. Early diagnosis with LFC in this group is highly recommended.38 Another risk factor associated with female cryptococcosis is systemic lupus erythematous or other autoimmune diseases, as it was seen in 2 of our patients.75 Other risk factors to be considered are diabetes and corticosteroids, as it was observed in this group and also in 5/6 cases in an intensive care unit in Arkansas.44,70 This seems to be also a predisposing factor in HIV-positive individuals according to a previous study.50

Based on the data obtained through 30 years, it can be concluded that meningoencephalitis has been the predominant clinical form, frequently with few symptoms (headache and fever as the most important ones) in cryptococcosis. C. neoformans (genotype VNI) has been isolated in most of the cases. On numerous occasions the blood culture, as well as skin or respiratory cultures, made the diagnosis possible.10,46,57,58 The use of sunflower agar in primary cultures has been of great help in these cases. Those HIV-positive individuals with CD4+ counts <100 cells/μl are at risk for this mycosis, and CrAg detection using the FLC technique in serum can be of great help for an early diagnosis.72 In Argentina, as in many countries, 5FC is not available and in the Muñiz Hospital the initial treatment with the AMB+FCZ combination has proved to be efficient. Along with a correct management of high intracranial pressure, especially in the first days, mortality rate diminished and results are encouraging. The failure in the treatment is probably due to a combination of causes more than an increased resistance to the antifungal drugs.1,74

The authors declare no conflict of interest.