Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 3620
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size

  Table of Contents    
Year : 2017  |  Volume : 60  |  Issue : 2  |  Page : 236-238
Sporadic occurrence of cryptococcal meningitis in HIV-seronegative patients: Uncommon etiology?

1 Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, New Delhi, India
2 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India

Click here for correspondence address and email

Date of Web Publication19-Jun-2017


Cryptococcosis in HIV-seronegative patients is rarely reported from India. This prospective study was conducted to look for cryptococcal meningitis in HIV-seronegative individuals and compare their laboratory features to cryptococcal meningitis in HIV-seropositive patients. Cerebrospinal fluid was collected from 153 suspected cases of meningitis and subjected to India ink preparation, antigen detection, and culture. Nineteen samples tested positive for Cryptococcus neoformans infection. Seventeen and two patients were HIV reactive and nonreactive, respectively. In vitro susceptibility of C. neoformans isolates to fluconazole and amphotericin B was performed using standard broth microdilution method and E-test. Eighteen strains were susceptible to amphotericin B, while fluconazole was reported susceptible in 15 strains. Hence, index of suspicion of C. neoformans infection as possible cause of meningitis must be maintained even in HIV-negative patients. Use of amphotericin B for treating C. neoformans meningitis should be restricted to prevent any increase in resistance.

Keywords: Amphotericin B, C. neoformans, fluconazole, HIV, meningitis

How to cite this article:
Das S, Datt S, Roy P, Saha R, Xess I. Sporadic occurrence of cryptococcal meningitis in HIV-seronegative patients: Uncommon etiology?. Indian J Pathol Microbiol 2017;60:236-8

How to cite this URL:
Das S, Datt S, Roy P, Saha R, Xess I. Sporadic occurrence of cryptococcal meningitis in HIV-seronegative patients: Uncommon etiology?. Indian J Pathol Microbiol [serial online] 2017 [cited 2022 Jan 22];60:236-8. Available from: https://www.ijpmonline.org/text.asp?2017/60/2/236/208402

   Introduction Top

Cryptococcus neoformans is an encapsulated yeast-like organism causing infections worldwide.[1] Inhalational route is the major route of transmission of cryptococcosis besides local transmission through skin and eyes.[2]C. neoformans causes human diseases ranging from asymptomatic colonization to severe meningitis and disseminated infection.[1] Nevertheless, central nervous system is the most common site of cryptococcal infection. Cryptococcal meningitis is the most fatal form of cryptococcosis.[3] Recent data indicate that the occurrence of cryptococcal infection is high in the developing nations including India.[3],[4] Cryptococcosis is generally found in association with acquired immunodeficiency syndrome (AIDS) although it has been reported to sometimes cause disease in HIV-seronegative patients as well.[1] There are very few reports of cryptococcosis in HIV-seronegative patients from India.[3],[5] Here, we report four cases of cryptococcal meningitis in HIV-seronegative individuals from a 1800-bed tertiary care hospital in Delhi and their laboratory diagnostic features in comparison to cryptococcal meningitis in HIV-seropositive patients.

   Materials and Methods Top

This was a prospective study conducted on cerebrospinal fluid (CSF) samples received in the Department of Microbiology at a 1800-bed tertiary care hospital in Delhi from clinically suspected cases of meningitis during a 3-year period from December 2012 to December 2015. Human ethical clearance was obtained from the institute. Written informed consent was taken from the study participants.

Four to five milliliters of CSF was collected from each patient. CSF was centrifuged at 2000 g for 1 min, and the supernatant stored at −20°C for antigen detection. The CSF sample was subjected to India ink preparation and rapid antigen detection. Antigen detection was done by using commercial kit, IMMY, CrAg lateral flow immunochromatographic assay (Germany). The CSF sample was also cultured following standard techniques on Sabouraud dextrose agar without cycloheximide but with antibiotics, along with birdseed agar, followed by incubation at 25°C.[6]

Antifungal susceptibility testing

In vitro susceptibility of C. neoformans isolates to fluconazole and amphotericin B was performed using the standard broth microdilution method as recommended by the CLSI M27-A3 protocol.[7] Quality control strains included were Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258. Fluconazole and amphotericin B were obtained as powder from HiMedia, Mumbai, and Sigma-Aldrich, Mumbai, respectively. Stock solutions of fluconazole and amphotericin B were prepared in sterile distilled water and dimethyl sulfoxide, respectively. Stock solutions were diluted with RPMI 1640 (RPMI tissue culture medium supplemented with glutamine) (Sigma-Aldrich, Mumbai) with 2% glucose, buffered to pH 7.0 with 0.156M 3-n-morpholinopropane-sulfonic acid (Sigma-Aldrich, Mumbai). The final concentrations were 0.12–64 μg/l and 0.013–16 μg/l for fluconazole and amphotericin B, respectively. The final inoculum concentration ranged from 0.5 ×103 to 2.5 ×103 cells/ml. Testing was performed in 96-well round-bottomed microtiter plates and was incubated at 35°C for 48–72 h. The minimum inhibitory concentration (MIC) of fluconazole and amphotericin B was read as the lowest concentration of the agent, which inhibited growth by 50% and 80%, respectively.

The range of antifungal dilutions used was as follows: fluconazole: 64–0.125 μg/ml and amphotericin B: 16–0.03 μg/ml. E-test for each isolate was also performed as per manufacturer's guidelines (HiMedia disk) against both amphotericin B and fluconazole. Control strains used were Candida albicans ATCC 24433, C. krusei ATCC 6258, C. parapsilosis ATCC 22019. The isolate was considered susceptible for amphotericin B if MIC was ≤1 μg/ml and for fluconazole if MIC was ≤8 μg/ml.

Clinical details were recorded and patients were provided treatment with fluconazole and amphotericin B if they tested positive.

Statistical analysis

Frequency distributions were obtained and percentages were calculated accordingly. GraphPad Inc. statistical software (2236 Avenida de la Playa, La Jolla, CA 92037, USA) was used for the calculation of P value using Fisher's exact test and one-sample t-test as applicable. Statistical significance was defined as P< 0.05.

   Results Top

A total of 153 CSF samples (31 females and 122 males) were collected from patients suspected to be suffering from meningitis. Nineteen (12.4%) were positive for C. neoformans infection by antigen detection, direct microscopic examination (India ink preparation), and culture. Age range and mean age of the 19 patients were 2–78 years and 34 years, respectively, with male predominance (18 males and 1 female), which was statistically significant (P = 0.00303). Of the 19 patients, 17 (89.5%) were HIV reactive and 2 (10.5%) were HIV nonreactive. Both HIV nonreactive samples belonged to male patients. Mortality rate was 42.11% (8 out of 19) in cases of meningitis.

Hematological and cerebrospinal fluid parameters

The mean total leukocyte count of the 19 study participants was 5478 cells/μL, which is within the normal range (4000–11,000 cells/μL). However, the mean hemoglobin level was much lesser at 8.51 g/dl and was statistically significantly (P < 0.0001) different from normal hemoglobin levels (13–16 g/dl), while mean blood urea level was raised (53.57 mg/dl) than the normal (15.2 mg/dl). CSF analysis revealed normal protein levels (mean protein = 42.67 mg/dl) and low glucose (mean glucose = 35 mg/dl; normal CSF glucose = 45–80 mg/dl, P= 0.0001) levels.

Susceptibility of study isolates to antifungals

Eighteen (94.74%) strains were susceptible to amphotericin B (both by MIC and E-strip). Fluconazole was reported susceptible in 15 (78.95%), susceptible but dose-dependent in 3 (15.79%), and resistant in 1 (5.26%) strain as per the CLSI guidelines [Table 1]; however, by E-strip, all strains were in the susceptible zone. Both HIV nonreactive samples were susceptible to fluconazole and amphotericin B, while among the HIV reactive samples, 13 (76%) and 16 (94%) were susceptible to fluconazole and amphotericin B, respectively.
Table 1: Minimum inhibitory concentration values for amphotericin B and fluconazole of the 19 study participants suff ering from Cryptococcus neoformans infection

Click here to view

   Discussion Top

The present study shows that though the number of new HIV infections has dropped drastically from 2.51 lakhs in 2000 to 86,000 in 2015, C. neoformans still remains the primary opportunistic infection in AIDS patients.[8] Most of the cases were in the age group of 28–50 years. This is in accordance with the national level statistics published by the National AIDS Control Organisation (NACO) in 2015.[8]

Males predominated the study group in statistically significant numbers. This finding is also in agreement with the national level statistics published by NACO in 2015.[8] Disparity in the gender distribution may be due to difference in exposure and not in susceptibility.[9] However, in a few instances, females have also been found to be affected with cryptococcal meningitis more often than males.[1]

The study participants had low blood hemoglobin levels, which was statistically significant and is a known feature of persistent cryptococcal meningitis.[10] Blood urea was also raised in them which is another recognized feature of cryptococcal meningitis.[11] Hypoglycorrhachia, a known poor prognostic marker of cryptococcal meningitis, was seen in most of the patients in statistically significant levels, which is similar to the previous studies.[11],[12]

Resistance to fluconazole was encountered in almost one-fourth of HIV-seropositive individuals, while amphotericin B resistance was detected at a much lower level (6%), conforming to prior studies.[13] Amphotericin B, fluconazole, and flucytosine are used in the treatment of cryptococcal meningitis, irrespective of coexisting HIV status.[3] Flucytosine is not commonly used in India because of its unavailability and high price.[3],[12] Combination of amphotericin B and flucytosine is recommended initially for the first 2 weeks, after which fluconazole is started.[11],[14] Resistance of C. neoformans to fluconazole and amphotericin B has been reported in previous studies as well.[15] Nonetheless, unlike fluconazole-resistant candidiasis, fluconazole-resistant cryptococcosis is not widely recorded.[15] The present study adds to the limited number of studies on this aspect. Surfacing of resistance to fluconazole suggests the need for alertness and extensive surveillance of chemosensitivity of clinical isolates at regular intervals.[3] As resistance to amphotericin B still has not reached dangerous proportions, its use must be restricted to prevent any further increase. Since cryptococcal meningitis is usually considered rare in immunocompetent individuals, specific treatment is not administered until the organism is identified or cryptococcal antigen is detected from the specimen.[3] Hence, despite the availability of newer antifungal agents, cryptococcosis in HIV-negative patients is often associated with considerable morbidity and mortality.[3]

   Conclusions Top

Our study demonstrates that C. neoformans should be suspected as a possible cause of meningitis even in HIV-negative patients. Resistance to amphotericin B was seen in much lesser number of isolates compared to fluconazole. Hence, use of amphotericin B for treating C. neoformans infection must be limited to avert further rise in resistance. Early diagnosis of cryptococcal meningitis and initiation of appropriate therapy are imperative as it will lead to improved clinical outcome and a better prognosis with lesser relapse rate.

Financial support and sponsorship

Financial assistance was provided to us by the Department of Biotechnology for conducting this research work.

Conflicts of interest

There are no conflicts of interest.

   References Top

Kiertiburanakul S, Wirojtananugoon S, Pracharktam R, Sungkanuparph S. Cryptococcosis in human immunodeficiency virus-negative patients. Int J Infect Dis 2006;10:72-8.  Back to cited text no. 1
Okwara FN, Makewa S, Karo E. Cryptococcal meningitis in a none-HIV infected five month old infant with rickets: Case report. East Cent Afr Med J 2015;2:106-8.  Back to cited text no. 2
Gupta P, Malik S, Khare V, Banerjee G, Mehrotra A, Mehrotra S, et al. A fatal case of meningitis caused by Cryptococcus neoformans var. grubii in an immunocompetent male. J Infect Dev Ctries 2011;5:71-4.  Back to cited text no. 3
Chakrabarti A, Sharma A, Sood A, Grover R, Sakhuja V, Prabhakar S, et al. Changing scenario of cryptococcosis in a tertiary care hospital in North India. Indian J Med Res 2000;112:56-60.  Back to cited text no. 4
Khanna N, Chandramuki A, Desai A, Ravi V, Santosh V, Shankar SK, et al. Cryptococcosis in the immunocompromised host with special reference to AIDS. Indian J Chest Dis Allied Sci 2000;42:311-5.  Back to cited text no. 5
Chander J, editor. Textbook of Medical Mycology. 3rd ed. New Delhi, India: Mehta Publishers; 2010.  Back to cited text no. 6
Clinical and Laboratory Standards InstituteM27-A3. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 3rd ed. Wayne, PA, USA: CLSI Document; 2008. .  Back to cited text no. 7
India HIV Estimations 2015 Technical Report. National AIDS Control Organisation (NACO); 2015. Ministry of Health and Family Welfare, New Delhi, Government of India.  Back to cited text no. 8
Banerjee U. Progress in diagnosis of opportunistic infections in HIV/AIDS. Indian J Med Res 2005;121:395-406.  Back to cited text no. 9
Xu XG, Pan WH, Bi XL, Fang W, Chen M, Zhu Y, et al. Comparison of clinical features in patients with persistent and nonpersistent cryptococcal meningitis: Twelve years of clinical experience in four centers in China. CNS Neurosci Ther 2013;19:625-31.  Back to cited text no. 10
Kumar S, Wanchu A, Chakrabarti A, Sharma A, Bambery P, Singh S. Cryptococcal meningitis in HIV infected: Experience from a North Indian tertiary center. Neurol India 2008;56:444-9.  Back to cited text no. 11
[PUBMED]  [Full text]  
Satishchandra P, Mathew T, Gadre G, Nagarathna S, Chandramukhi A, Mahadevan A, et al. Cryptococcal meningitis: Clinical, diagnostic and therapeutic overviews. Neurol India 2007;55:226-32.  Back to cited text no. 12
[PUBMED]  [Full text]  
Pan W, Khayhan K, Hagen F, Wahyuningsih R, Chakrabarti A, Chowdhary A, et al. Resistance of Asian Cryptococcus neoformans serotype A is confined to few microsatellite genotypes. PLoS One 2012;7:e32868.  Back to cited text no. 13
Bicanic T, Harrison TS. Cryptococcal meningitis. Br Med Bull 2005 18;72:99-118.  Back to cited text no. 14
Cheong JW, McCormack J. Fluconazole resistance in cryptococcal disease: Emerging or intrinsic? Med Mycol 2013;51:261-9.  Back to cited text no. 15

Correspondence Address:
Priyamvada Roy
Flat No. C-115, Jalvayu Vihar (Near AWHO), Plot No. 8, Pocket P-4, Greater Noida - 201 310, Uttar Pradesh
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_599_16

Rights and Permissions


  [Table 1]

This article has been cited by
1 Global HIV neurology
Kiran T. Thakur,Alexandra Boubour,Deanna Saylor,Mitashee Das,David R. Bearden,Gretchen L. Birbeck
AIDS. 2019; 33(2): 163
[Pubmed] | [DOI]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  

    Materials and Me...
    Article Tables

 Article Access Statistics
    PDF Downloaded116    
    Comments [Add]    
    Cited by others 1    

Recommend this journal