C. neoformans is a major cause of disease in immunocompromised hosts, especially in people living with HIV/AIDS, with approximately one million cases globally of cryptococcal meningitis each year.¹Some studies have shown that up to 10-40% of cases of cryptococcosis occur in otherwise immune competent patients.² Diagnosis and treatment guidelines for cryptococcosis in immune competent patients are based predominantly on data derived from the HIV infected population. A few small studies from India have described cryptococcal meningitis in apparently immune competent hosts.^3,4

We aimed to study and compare the clinical presentation, diagnosis, progress and outcome of cryptococcal disease in apparently immune competent and immunocompromised hosts.

We conducted an observational retrospective study by reviewing medical case records of adult patients with any form of cryptococcal disease admitted from June 2009 to June 2015 at a 700-bedded tertiary care referral hospital in South India. Cases were identified through the hospital’s computerized Clinical Management System (by performing a search for the ICD-9 code for "cryptococcosis” or "cryptococcal meningitis”) and the microbiology laboratory. Medical records were analyzed for demographic details, clinical presentation, underlying immune compromising conditions such as HIV infection, organ transplant, cancer, cirrhosis, end-stage chronic kidney disease, autoimmune diseases, diabetes mellitus and chronic administration of corticosteroids. Patients without any of these potential predisposing factors were defined for the purpose of this study as immune competent.

Data of cerebrospinal fluid (CSF) analysis (white blood cell (WBC) and differentials, glucose levels, protein levels, India ink stain, cryptococcal antigen and culture) were collected from the case records. Similarly, data on culture and cytology/histopathology data on pus, lymph node biopsy and/or biopsy or aspiration from other tissues was retrieved whenever available.

Details on antifungal treatment and duration were collected. Drugs used included conventional or liposomal amphotericin B, accompanied or not by flucytosine, followed by fluconazole, or fluconazole alone.

For the purpose of this study, we defined the clinical outcomes as "unsatisfactory” if death occurred during hospitalization or if a relapse (microbiologically confirmed) occurred within 2 months of discontinuing antifungal therapy. Patients were monitored at discharge, at 3 months after treatment commencement, at one year after treatment commencement, and at 6 months after completing maintenance treatment. A "satisfactory” outcome at discharge was defined as survival during the admission period and a "satisfactory” outcome at follow up was defined as no relapse during 6 months after stopping antifungal therapy.^2,5,6

Microbiological investigations

Specimens were inoculated on Sabouraud dextrose agar plates, for fungal culture. Following isolation of yeasts from normally sterile sites, we performed identification of Cryptococcus using a combination of CHROMagar (Paris, France), microscopic morphology and the VITEK system (bioMérieux, Marcy-l'Étoile, France). The cryptococcal antigen lateral flow assay was performed for cryptococcal antigen detection.

Statistical methods

Continuous data were expressed as mean±standard deviation (SD) or as median (interquartile range), according to their distribution. Categorical variables were analysed with the Chi square test or Fisher’s exact test (two-tailed). Continuous variables were compared by using Student’s t test. A p value of <0.05 was considered statistically significant.

A total of 20 patients with cryptococcal disease were identified, of whom 8 (40%) were immune competent while 12 patients were immunocompromised. Out of 12 immunocompromised patients, 10 were HIV positive, one patient had a post renal transplant status and one patient had chronic liver disease and diabetes. Details are shown in Table 1.

Table 1. Demographic details, comorbidities and characteristic of organ involvement in the two study groups

A total of 16 patients had cryptococcal meningitis while 4 patients had extra central nervous system (CNS) disease (Table 1). In the immunocompromised group, all (n=12) patients presented with meningitis while in the immune competent group, 4 patients presented with meningitis and 4 patients with extra CNS disease (without clinical evidence of CNS involvement) (p=0.014).

There was no statistically significant difference in presenting complaints between the two groups (Table 2). However the median time to diagnosis was much longer in immune competent patients compared to immunocompromised patients, 90 (15-110) days and 20 (7-75) days, respectively (p=0.01).

CSF findings were similar (Table 2) in both groups except that mean CSF protein levels were significantly lower (78.5±15.1 mg/dL) in immune competent patients compared to immunocompromised patients (181.9±150.9 mg/dL) (p=0.036). CSF cryptococcal antigen was positive in both groups, in all patients with meningitis. Data on CNS imaging is presented in Table 2 for all patients with meningitis.

 

Table 2. Clinical presentation, CSF parameters and CNS imaging characteristics in the two study groups

 

In four patients with extra CNS disease, diagnosis was made on histopathology examination of various samples, while fungal cultures were available for only 2 patients. Serum cryptococcal antigen was available for 5 patients in the immune competent group and 9 patients in the immunocompromised group: all were positive. Routine CSF examination was not performed for immunocompetent patients with extra CNS cryptococcosis.

Antifungal therapy included conventional or liposomal amphotericin B, accompanied or not by flucytosine, or fluconazole alone (Table 3). The majority of patients received conventional amphotericin B 0.7 mg/kg with flucytosine 100 mg/kg for a mean duration of 2 weeks in immunocompromised hosts and 3 to 4 weeks in the immune competent group. Patients in both groups received fluconazole maintenance treatment for 1 year. Two patients in the immunocompromised group and 2 patients in the immune competent group required multiple therapeutic lumbar punctures followed by CSF shunt to manage raised intracranial tension during hospitalization. One patient with meningitis from the immune competent group had persistently positive CSF India ink for cryptococci and cryptococcal antigen; he had received 2 courses of 4 weeks of conventional amphotericin B and ultimately died 6 months following diagnosis, although all follow up CSF cultures were negative. Two patients in each group took discharge against medical advice after 2 days of treatment, and were presumed to have had a poor outcome. A total of 5 (83.3%) patients in the immune competent group and 9 (100%) patients in the immunocompromised group had satisfactory outcome at the time of discharge.

We assessed outcome at 3 months and 1 year following discharge and at 6 months after completion of antifungal therapy: 4 patients were lost to follow up and 1 patient in the immunocompetent group showed persistently positive India ink examination but negative cultures and required multiple shunt surgeries and ultimately died because of shunt related complications. One (16.7%) patient in the immunocompetent group and 1 (11.1%) patient in the immunocompromised group developed recurrence; the patient in the immune competent group had defaulted treatment and the patient in the immunocompromised group experienced relapse 2 years after completion of treatment. All other patients had satisfactory outcome during treatment and at 6 months of follow up after completing treatment.

 Table 3. Treatment details and outcome of patients in both study groups

The tropical climate found in the Indian subcontinent represents a suitable milieu for Cryptococcus neoformans.⁷ Risk factors for cryptococcosis include HIV infection, use of immunosuppressive agents, and the existence of predisposing comorbidities such as hematologic disorders, organ failure syndromes, innate immunologic deficits, or organ transplant.¹

Data on the epidemiology, treatment and subsequent outcome of cryptococcal infections in immune competent patients are scarce. All the available data are either isolated case reports or small case series.^2-6,8-10Eight patients (40%) in our study were immunocompetent, which is comparable to the study by Prasad et al.³ and the review by Pappas et al. (10-40%)²while the incidence was lower (12.8%) in the study by Abhilash et al.³

The most common presenting complaint in our study was headache (70%) which is concordant with other studies from India.^3,4Fever (50%) and altered sensorium (35%) were next, similar to the study by Abhilash et al (75% and 41% respectively).³ None of the patients in our study had seizures at presentation or during treatment, while other studies reported a seizure incidence of 10.5% and 18%.^3,4

The time to diagnosis was significantly higher in the immunocompetent group (90 days versus 20 days), concordant with other studies.^2-5 This might be related to the lower index of clinical suspicion as there were no statistically significant differences in clinical presentation between these two groups. In our study, we found more extra CNS involvement in the immunocompetent group compared to the immunocompromised group (4 vs. 0, p=0.014), which is not concordant with other studies.^4,5

While two major studies^4,5 found a significantly raised CSF cell count in the immune competent group compared to the immunocompromised group, in our study we did not find a statistically significant difference between the two groups (p=0.188), which is comparable to the study by Abhilash et al.³ In our study, the mean CSF protein was significantly higher in the immunocompromised group (p=0.036) which is not concordant with other studies.^2-5 There was no significant difference in CSF glucose (p=0.142) which is concordant with other studies.^2,4,5Serum cryptococcal antigen was positive in all patients in whom the test was ordered and serves as a valuable screening test, concordant with guidelines for HIV infection.¹¹

In the absence of randomized controlled trials, recommendations for the treatment of cryptococcosis in the immune competent group are based on isolated case reports and case series.^3,6,9,12,13 In our study two patients in each group developed raised intracranial tension requiring multiple therapeutic lumbar punctures and shunt placement, unlike in the study by Pappas et al.² where this complication was higher in the immunocompetent group. There was not a significant difference between outcome at discharge and recurrence between the two groups in our study while other studies^2,5 demonstrated better outcome in an immunocompetent group as compared to an immunocompromised group.

The major limitation of our study was the inclusion of only 20 patients, limiting our ability to perform an extensive analysis.

We have shown that cryptococcal disease is not uncommon in immune competent hosts and diagnosis is usually delayed because of a low index of clinical suspicion. Serum cryptococcal antigen could serve as a valuable screening test, and in our study it was positive in all our patients. Our other findings of a preponderance of extra CNS disease and a lower CSF protein level in immune competent patients require confirmation in a larger cohort. Despite the fact that we found no significant difference in the outcome between the two groups, the median time to diagnosis was much longer in the immune competent group compared to the immune compromised group, which emphasizes the need for clinical awareness of this entity in the immune competent group.

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