Prevalence of Cryptococcal Meningitis among Immuno-competent and Immuno-compromised Individuals in Bellary, South India: A Prospective Study

1. Corresponding Authors, 2. Assistant Professor, Dept of Microbiology, S.D.M.Medical college, Dharwad, India 3. Professor and Head, Dept of Neuromicrobiology, NIMHANS,Bangalore, India. 4. Professor,Dept of Medicine, VIMS,Bellary, India. 5. Assistant Professor, Dept of Paediatrics, Srinivas Institute of Medical Sciences and research centre, Surathkal Vijayanagar Institute of Medical Sciences, Bellary, India.

Abstract

Background and Objectives: Cryptococcal meningitis is now the leading cause of community acquired meningitis. It has generally been thought to be associated with individuals with AIDS. However, other predisposing factors like leukaemia, lymphoma, Diabetes mellitus, connective tissue disorders and organ transplantation also contribute to this infection. Cryptococcal meningitis also occurs in apparently immuno-competent individuals. Opportunistic infections are the major life threatening complications of the acquired immuno-deficiency syndrome (AIDS). An early diagnosis can help the clinician to treat cryptococcal meningitis and to thus help in reducing the rate of mortality. The present study was taken up to evaluate the occurrence of cryptococcal meningitis among immuno-compromised and immuno-competent individuals and also to analyze the predisposing factors which contribute to its occurrence.

Methods: A total of 242 CSF samples of clinically suspected cryptococcal meningitis were screened for Cryptococcus neoformans irrespective of their immune status. Their identification was based on direct microscopy, culture and biochemical reactions which were carried out by conventional methods. The CD4 cell count was obtained by flow cytometry in all the culture positive patients.

Results: The overall prevalence of cryptococcal meningitis was 8.3% and the prevalence of cryptococcal meningitis among the immuno-compromised patients was found to be 16.6%. 60% of the patients were in the age group of 21-40 years. Infection with HIV (100%) was the most common predisposing factor, followed by diabetes mellitus (40%), chronic smoking (20%) and prolonged steroid therapy (5%). Cryptococcus neoformans var. neoformans was the aetiologic agent in all the culture positives in our study. Cryptococcal meningitis was the AIDS defining illness in 50% of the patients. The mean CD4 count was 59.55. 65% of the patients had a CD4 count of <100.

Conclusion: The high prevalence of cryptococcal CNS infections in HIV infected patients underscores the importance of a precise and early microbiological diagnosis. A high index of clinical suspicion and mycological surveillance is required to help in an early diagnosis and appropriate therapy.

Cryptococcal meningitis, AIDS, Immunocompromised,Cryptococcus neoformans, CNS infections

Introduction
Cryptococcosis was considered as a clinical rarity in the early 1900s (1). The number of cases of cryptococcosis has dramatically increased since the 1950s. This increase has resulted partly from the greater awareness about Cryptococcus neoformans as a human pathogen, but mostly it parallels the ever-enlarging population of immuno-compromised individuals as a result of aggressive chemotherapies, organ transplantations and the acquired immunodeficiency syndrome (2). Cryptococcus neoformans is the second most common lethal fungal opportunist after Candida albicans, which causes symptomatic cryptococcosis in 6–13% of the patients with AIDS (1),(4),(2),(6). C. neoformans, the causative organism, is ubiquitous in distribution. A vast majority of the isolates which are responsible for infections are of the C. neoformans var. neoformans serotype A (3). The organism gains access into the host usually via the respiratory route and it is generally contained there by an intact cell mediated immune system. However, in the presence of immunodeficiency, C. neoformans disseminates widely especially to the central nervous system (CNS) (4). It has a special propensity for invading the CNS. Meningitis is the commonest CNS manifestation of a cryptococcal infection (4). It presents as a sub-acute or chronic illness without overt meningeal disease or encephalopathy (5). The diagnosis is established by CSF analysis which demonstrates the organism on India ink preparation or culture. Cryptococcal meningitis is an important fatal infection of the CNS, wherein, the signs and symptoms are indistinguishable from those of other sub acute and chronic CNS infections. An early diagnosis can help the clinician to treat cryptococcal meningitis and it thus helps in reducing the mortality rate. It is generally thought to be associated with individuals with AIDS. However, other predisposing factors like leukaemia, lymphoma, diabetes mellitus, non-insulin dependent diabetes mellitus (NIDDM), connective tissue disorders and organ transplantation also contribute to the infection (4). Cryptococcal meningitis also occurs in apparently immunocompetent individuals (4).

The perceived importance of C. neoformans as a human pathogen, has led to the species being subjected to intense study during the last decade. The present study was taken up to study the occurrence of cryptococcal meningitis in immunocompromisedand immunocompetent individuals in the Bellary region of south India. The patient’s demographic data, the predisposing factors for cryptococcal meningitis, the risk factors for acquisition of the HIV infection, AIDS defining conditions, other concurrent infections, the presenting clinical symptomatology, physical findings, laboratory parameters and the cerebrospinal fluid (CSF) examination findings were analyzed in relation to its occurrence.

Material and Methods

This study was carried out in the Department of Microbiology, Vijayanagar Institute of Medical Sciences, Bellary, India from December 2007 – December 2008. The catchment area of the patients who attended this hospital was predominantly the Bellary district and the neighbouring districts. A total of 242 CSF samples were processed during the study period. Clinically suspected cryptococcal meningitis cases, irrespective of their immune statuses were included in the study. Meningitis cases which were caused by other aetiologies were excluded from the study. All the CSF samples were collected before instituting the therapy. The samples were considered as a potential biohazard and they were handled with care by using universal precautions. A haemogram and a CD4 cell count was perfomed in all the patients who were involved in the study. The samples were subjected to the evaluation of their cell count and cell types, proteins and sugar, direct microscopy by the India ink preparation, gram staining and culture for speciation and biotyping.

The CSF samples were inoculated on duplicate sets of Sabouraud’s dextrose agar (SDA) slopes which were devoid of cycloheximide for the culture. They were incubated at 25oC and 37oC, separately over a period of four weeks. The cultures which yielded smooth, cream-buff coloured, moist and mucoid colonies at 37oC were considered as positive. The suspected colonies on SDA were confirmed by doing gram staining and India ink preparations. The following biochemical tests were performed for the speciation of the isolates. A. The Urease test: The Urease test was performed by using Christensen’s urea agar with a phenol red indicator (6). B. The Inositol assimilation test: It was performed by using a yeast nitrogen base with a bromocresol purple indicator (7). C. The Test for phenol-oxidase: It was performed by using Staib’s bird seed agar (8).

Biotyping of the isolates was done by using Canavanine Glycine Bromothymol blue (CGB) agar. The CGB agar was prepared according to Kwon Chung et al’s method (9). The CD4 cell count was obtained by flow cytometry, by using a BD FACScount machine. Cryptococcal meningitis was diagnosed by checking for the clinical features of meningitis/meningoencephalitis and by checking for a positive CSF India ink preparation and/or the isolation of C. neoformans in the CSF culture. Patients who presented with the clinical features of meningitis/meningoencephalitis of less than four weeks duration were defined as having an acute or a subacute presentation. The following criteria were used to define the common concomitant infections: Pulmonary tuberculosis (PTB): The clinical features which were suggestive of TB with radiological features were compatible with TB on chest radiographs or computed tomographic (CT) scans, the demonstration of acid-fast bacilli (AFB) in the sputum smears or the growth of Mycobacterium tuberculosis in the sputum cultures. Pneumocystis jiroveci pneumonia (PCP): Bilateral, diffuse interstitial infiltrates on chest radiographs or high-resolution CT, with hypoxaemia (PaO2 <12 kPa), sputum smears/cultures which were negative for aerobic bacteria and AFB and/or the demonstration of Pneumocystis jiroveci in the induced sputum.

A descriptive statistical analysis was carried out in the present study. The results of the continuous measurements were presented as mean = SD (min-max) and the results of the categorical measurements were presented as percent. The significance was assessed at a 5% level of significance. The prevalence of Cryptococcal meningitis (culture positivity) was estimated and its prevalence was correlated according to the age, gender, marital status, partner status, CSF analysis and the CD4 count analysis of the patients by using the single proportion test. The diagnostic statistics viz: sensitivity, specificity, positive predictive value, negative predictive value and accuracy were computed for evaluating the India ink method results against the culture positivity. The statistical softwares, namely, SPSS 15.0, Stata 8.0, MedCalc 9.0.1 and Systat 11.0 were used for the analysis of the data and Microsoft word and Excel were used to generate the graphs and the tables.

Results

The study population consisted of 242 patients, of which 168 were males and 74 were females. Of the 242 patients, 122 patients were immunocompromised and 120 of them were immunocompetent. The demographic profile of the study population is shown in (Table/Fig 1). Among the 242 patients, C. neoformans var. neoformans was isolated in 20 patients. Twenty patients who were diagnosed to have cryptococcal meningitis were analyzed. All of them had a compromised immune status. Cryptococcal meningitis was not prevalent in the immunocompetent patients. The immunocompromised state was significantly associated with the culture positivity (Table/Fig 2). The overall prevalence of cryptococcal meningitis in the study population was 8.3% and that in the immunocompromised group was 16.6%. The mean age of presentation was 28.6+6 years. Most of the patients (95%) were in the age group of 21-40 years (Table/Fig 1). None of the patients were above the age of 40 years. Among the culture positives, 55% of the patients were males. The male: female ratio was 1.2:1.

Infection with HIV (100%) was the most common predisposing factor, followed by chronic smoking (50%), diabetes mellitus (40%) and prolonged steroid therapy (5%) (Table/Fig 3). Cryptococcal meningitis was the initial presenting illness of an HIV seropositive status in 50% of the patients. Two (10%) patients had associated pulmonary tuberculosis, two (10%) had oral candidiasis, two (10%) patients had bronchopneumonia (Pneumocystis jeruveci) infection and one (5%) patient had herpes zoster infection. An identifiable risk factor for the HIV infection was present in 11 (55%) patients. Eleven (55%) patients were from the high risk groups, which included truck drivers and intravenous (IV) drug abusers. The commonest mode of transmission was multiple, heterosexual, unprotected sexual contact, which was seen in nine patients. One patient each had a history of unsafe blood transfusion and intravenous drug abuse.

Headache was the most common presenting symptom. It was present in 85% of the patients. In all the patients who had headache, it was found to be of severe intensity and it was situated bi frontally. Vomiting was the second most common presenting symptom, and it was seen in 80% of the patients. Low grade fever was present in 60 % of the patients. The other presenting symptoms included an altered sensorium (40%), seizures (20%) and motor deficits (10%). Most of the patients (75%) had symptoms of less than fourweeks duration (acute – subacute); 25% of patients presented with chronic meningitis. The CD4 cell count in our study ranged from 10 to 156 cells/μl. The median CD4 cell count of the whole group was 59.55 cells/μl. Twenty per cent of the patients had a CD4 count of less than 50 cells/dl and 65% of the patients had a CD4 count of less than100cells/dl.

Culture was taken as the gold standard against which the India ink preparation was compared. The sensitivity, specificity, positive predictive value, negative predictive value and the accuracy of the India ink preparation were 90%, 100%, 100%, 99.1% and 99.2% respectively. (Table/Fig 4), (Table/Fig 5). The India ink preparation of CSF was positive in 90 % of the patients. The cryptococcal culture of CSF was positive in all the 20 patients. All the positive cultures yielded C. neoformans var. neoformans. Since Cryptococcal meningitis was prevalent only in immunocompromised patients in our study, a comparison could not be done between the two groups.

Discussion

The prevalence of cryptococcal meningitis varies world wide, ranging between 6-13% in patients with AIDS (1),(2),(10). In the present study, cryptococcal meningitis was seen predominantly in immunocompromised individuals. It was not prevalent in immunocompetent patients. Cryptococcal meningitis is generally considered to be rare in immunocompetent patients and the diagnosis is often delayed because of the presence of non-specific symptoms. The overall prevalence of cryptococcal meningitis in the present study was 8.3%, which was similar to the findings of the studies which were done elsewhere in India and abroad (11),(12). Cryptococcal meningitis may affect persons of any age. However, the average age of the patients ranges from 30-40 years. In the present study, the patients had a mean age of 28.6+6 years, with 95% of the patients being in the age group of 21-40 years, the most productive age group in the country. This was in consonance with the technical report which provided the national level statistics, which was published by the National AIDS Control Organization (NACO) in the year 2006.

Cryptococcal meningitis is more frequently reported in men than in women and rarely in children (13). This may reflect a difference in the exposure rather than a difference in the susceptibility. In the present study, the number of males was more as compared to the number of females (1.2:1). This is in accordance with the findingsof other studies, which had demonstrated a higher prevalence of cryptococcal meningitis in men (14) In the present study, while the males belonged to a wider age spectrum, the females were of a considerably younger population, and most of them had acquired the infection from their spouses, thus reflecting the male dominance in the Indian society. This emphasizes an increased need for awareness and counseling of both the spouses. Cryptococcal meningitis is generally thought to be associated with individuals with AIDS. However, other predisposing factors like leukaemia, lymphoma, diabetes mellitus, connective tissue disorders and organ transplantation may also contribute to the infection. In the present study, cryptococcal meningitis was prevalent only in immunocompromised patients; the infection with HIV being predominant, followed by diabetes mellitus, chronic smoking and prolonged steroid treatment. In the present study, among the 20 culture positives, all the 20 patients were found to be HIV seropositive; of which 50% of the patients had been found to be HIV seropositive during prior admission for pulmonary tuberculosis, oral candidiasis and herpes simplex infection. Cryptococcal meningitis was the first manifestation of AIDS in 50% of the patients. In other words, cryptococcal meningitis was the AIDS defining illness in 50% of the patients. This finding is almost similar to that of the study which was done by Chuck et al.,(5) wherein cryptococcal meningitis was the AIDS defining illness in 45.28% of the patients. In this study, two patients had associated pulmonary tuberculosis, one patient had oral candidiasis, two patients had bronchopneumonia and one patient had herpes zoster infection. This was in consonance with the findings of two large studies on HIV-infected patients which were reported from the Indian subcontinent (15),(16).

The triad of headache, fever and vomiting were the predominant symptoms, which were similar to the earlier reports in the literature (17),(18). Headache was the most common presenting symptom. It was present in 85% of the patients. In all the patients who had headache, it was found to be of severe intensity and it was situated bi frontally. Cryptococcal meningitis usually presents in the form of chronic meningitis. In the present study, more than half the patients had an acute to a subacute presentation. This emphasizes that even with an acute presentation along with the features of meningitis in HIV-infected individuals, the possibility of cryptococcal meningitis must always be considered and that the patients must be investigated accordingly. The clinical presentation of cryptococcal meningitis in an indolent manner with fever and headache, with absence of the signs of meningeal irritation in more than 80% of the cases, necessitates a high index of suspicion. The diagnosis of cryptococcal meningitis was based on the CSF culture. Among the 20 culture positives, the India ink preparation showed positive results in 18 patients. In one out of the 18 patients, the India ink staining showed numerous capsulated budding yeast cells in CSF. In other cases, we had to diligently search all the fields of the smear from the deposit before it could be considered as positive. The CSF India ink preparation was positive in 90% of the patients. The CSF culture was superior to the India ink staining. By considering culture as the gold standard, the sensitivity of the India ink preparation was found to be 90%. The India ink preparation is a simple procedure and it is used as a screening procedure in cases of cryptococcal meningitis. The positivity in the present study with respect to the India ink preparation was similar to that of the studies which were done by Imwidthaya et al.,(19) Darras-Joly et al., (20) Kovac et al. and Thakur et al (21). The CSF culture for C. neoformans is the diagnostic gold standard test.

A vast majority of the isolates which were responsible for the cryptococcal infection were of the C. neoformans var. neoformans serotype A (3). Cryptococcus neoformans var. neoformans was found to be the aetiologic agent in all the culture positives in our study. It was possible that exposure to the neoformans variety was more common than the exposure to the gattii variety. This was therefore an epidemiologic problem which was closely associated with the geographic topography which was specific to the ecological niches of these two varieties. The Neoformans variety was found in pigeon droppings. The CD4+ T cell count was the best indicator of the immediate state of immunologic competence and also the strongest predictor of the HIV-related complications in these patients. Cryptococcal meningitis was one of the AIDS defining illness in patients with a CD4 count which was less than 100/ml [21.] In one study (20), the average CD4 lymphocyte count in patients with AIDS and cryptococcosis was 46 cells/μl.The median CD4 cell count in this case series was 59.55 cells/μl.

Conclusion

In conclusion, the present study demonstrated the prevalence of cryptococcal meningitis only in immunocompromised individuals. It had an acute to a sub-acute presentation in a majority of the patients. The frank meningeal signs were less common in the isolated cryptococcal meningitis cases. Infection with HIV continues to be the most important predisposing factor for the development of CNS cryptococcosis, followed by chronic smoking, diabetes mellitus and prolonged steroid therapy. The occurrence of meningitis in patients with the HIV infection was most frequently due to opportunistic infections. The biotype which was responsible for cryptococcal meningitis in and around Bellary was C.neoformans var.neoformans. Cryptococcal meningitis was the AIDS defining illness in 50% of the patients. Cryptococcal infection remains the major opportunistic infection in HIV-infected patients with a CD4 cell count of <100 cells/μl. A high index of clinical suspicion and mycological surveillance is required to help in an early diagnosis and appropriate therapy.

DOI and Others

DOI: JCDR/2012/3959:1989

DECLARATI ON ON COMPETING INTERESTS:
No competing Interests.


Date of Submission: Aug 23, 2011
Date of Peer Review: Oct 15, 2011
Date of Acceptance: Mar 10, 2012
Date of Publishing: May 01, 2012