Prevalence of cryptococcal meningitis among people living with human immunodeficiency virus/ acquired immunodeficiency syndrome in a Tertiary Care Hospital, Southern Odisha, India
Published on: Mar 4, 2016
Transcripts - Prevalence of cryptococcal meningitis among people living with human immunodeficiency virus/ acquired immunodeficiency syndrome in a Tertiary Care Hospital, Southern Odisha, India
ISSN : 0976-9668
Vol 5, Issue 2, July 2014
Biology and Medicine
J Nat Sci. Biol. Med.
Prevalence of cryptococcal meningitis among
people living with human immunodeficiency virus/
acquired immunodeficiency syndrome in a Tertiary
Care Hospital, Southern Odisha, India
Departments of Microbiology, and 1Radiodiagnosis, Maharaja Krishna Chandra Gajapati Medical
College and Hospital, Berhampur University, Berhampur, Odisha, India
Address for correspondence:
Dr. Muktikesh Dash, Department of Microbiology, Maharaja Krishna Chandra Gajapati Medical College
and Hospital, Berhampur ‑ 760 004, Odisha, India. E‑mail: email@example.com
Objective: Cryptococcal meningitis (CM) caused by encapsulated opportunistic yeast Cryptococcus neoformans is an
important contributor to morbidity and mortality in people living with human immunodeficiency virus/acquired immunodeficiency
syndrome (PLHAs). Early diagnosis of such patients is the key to their therapeutic success. A retrospective study was conducted
to evaluate the clinical features, laboratory findings, and prevalence of CM among hospitalized PLHAs in a tertiary care setting.
Materials and Methods: A total of 112 clinically diagnosed CM patients were subjected to cerebrospinal fluid analysis and
tests for human immunodeficiency virus antibodies by the standard laboratory operating procedures. Results: Out of 112, 16
showed a definite diagnosis of C. neoformans with the prevalence of 14.3%. Males in the age group of 21‑40 years were most
commonly affected than females. The clinical manifestations observed were fever and headache (100%), followed by altered
sensorium (93.7%), neck stiffness (75%), and vomiting (62.5%). Overall, Cluster of differentiation 4 (CD4) T‑lymphocytes count
was <100 cells/µl except 1 case in which the CD4 T‑lymphocytes count was 137 cells/µl. No concomitant cryptococcal and
tubercular meningitis case was detected. All 16 patients responded initially to induction therapy of IV amphotericin B 1 mg/kg
and fluconazole 800 mg daily for 2 weeks. Subsequently, 4 (25%) patients were lost for follow‑up and 2 (12.5%) patients expired
during their hospital stay. Conclusion: As the clinical and radiological pictures of CM are often non‑pointing, routine mycological
evaluation is necessary for early definite diagnosis and subsequent initiation of appropriate therapy as the majority of patients
respond well to treatment if started early.
Key words: CD4 T‑lymphocytes, cryptococcal meningitis, Cryptococcus neoformans people living with human
immunodeficiency virus/acquired immunodeficiency syndrome
Cryptococcal meningitis (CM) caused by Cryptococcus
neoformans is an opportunistic fungal infection in human
immunodeficiency virus (HIV)‑seropositive patients.
This encapsulated yeast is found in soil contaminated with
bird droppings particularly from pigeons and chickens,
usually inhaled through lungs and remain dormant
for many years. Reactivation, which occurs primarily
among immunosuppressed individual such as people
living with human immunodeficiency virus/acquired
immunodeficiency syndrome (PLHAs), leads to infection
and most of which is meningitis. CM is a significant cause
of morbidity and mortality among PLHAs world‑wide.[3‑6]
Cryptococcus infect an estimated 1 million people and results
in approximately 625,000 deaths annually. It is the most
common central nervous system (CNS) fungal pathogen
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Journal of Natural Science, Biology and Medicine | July 2014 | Vol 5 | Issue 2 324
Dash, et al.: Cryptococcal meningitis among PLHAs
The clinical signs and symptoms of CM are indistinguishable
from those of many other causes of meningitis. This
infection is fatal without treatment. Therefore, rapid
recognition, diagnosis, and treatment are required to
decrease the mortality. Recent data indicate that the
incidence of C. neoformans is high among PLHAs in
developing countries like India.[12,13] A retrospective study
was carried out in a Tertiary Care Hospital, Southern
Odisha, India to evaluate the clinical features, laboratory
findings, and prevalence of CM among PLHAs.
MATERIALS AND METHODS
The study was conducted in the Department of Microbiology,
a Tertiary Care Hospital of South Odisha, India from January
2010 to June 2012. A total of 112 HIV‑seropositive patients,
clinically diagnosed as CM were included. A retrospective
cross‑sectional study of the medical records of these patients
and their clinical data were evaluated.
The cerebrospinal fluid (CSF) samples of 112 cases were
processed for fungal culture after preliminary screening
by microscopic examination, comprising of wet mount,
Gram’s staining and negative staining with 10% Nigrosin.
All the samples were inoculated on two sets of Sabouraud’s
dextrose agar (SDA) without cycloheximide, one incubated
at 37°C, another at 24°C, in special biological oxygen
demand incubator. The colony morphology was noted.
C. neoformans was identified base on yeast like mucoid
colony on SDA and urease test. For a definite diagnosis,
colony from SDA was sub‑cultured on Niger seed agar,
which was incubated at 37°C and observed for appearance
of brownish to blackish colored colonies due to melanin
pigment production by C. neoformans.
Written consent was sought before HIV testing was carried out.
All the serum samples were tested for HIV antibodies by three
rapid tests protocol as per the guidelines laid down by World
Health Organization (testing strategy III) and Government
of India. The CD4 T‑lymphocytes enumeration was
performed by using the BD FACS™ Calibor system (Becton
Dickinson, Fluorescent antibody cell sorter, Singapore). All the
tests were performed in accordance with the Medical College
Institutional Ethical Committee guidelines.
The values of mean, and P value were calculated by using
the GraphPad statistical software. Statistical significance
was defined when P < 0.05.
From 112 processed CSF samples, 16 yielded growth of
C. neoformans, showing prevalence of 14.3% [Table 1].
Out of all laboratory confirmed CM cases, 12 (75%) were
male and 4 (25%) were female. Most of the CM positive
patients were in the age group of 20‑40 years (81.3%)
ranging from 22 years to 53 years of age with a mean of
35.1 years [Table 2]. Out of 16 patients, 3 (18.7%) were
aware of their HIV status, but subsequently did not came
for antiretroviral therapy (ART) counseling and follow‑up,
10 (62.5%) were diagnosed only after hospital admission
and three (18.7%) were on ART.
The clinical presentations were almost same in most of the
patients showing features of meningitis. Most common
signs and symptoms being fever and headache (100%),
followed by altered sensorium (93.7%), neck stiffness (75),
vomiting (62.5%), and wasting (56.2%) [Figure 1]. The
CSF cell counts, glucose, and protein concentrations were
Preliminary microscopic examination by negative staining
with 10% Nigrosin could identify 13 (81.3%) cases of
Table 1: Year wise distribution of CM in PLHAs
Year Total clinically
2010 37 05 P=1.000 (not
2011 48 07
2012* 27 04
Total 112 (100) 16 (14.3)
CM: Cryptococcal meningitis,*Data up to June 2012, P ≤ 0.05 (statistically
significant), PLHAs: People living with HIV/AIDS
Table 2: Age and sex wise distribution of clinically suspected and laboratory confirmed CM in PLHAs
Age group in years Clinically suspected CM cases (n=112) Laboratory confirmed CM cases (n=16)
Male Female Total (%) Male Female Total (%)
0‑10 02 01 03 (2.7) 0 0 0
11‑20 03 02 05 (4.5) 0 0 0
21‑30 25 11 36 (32.1) 04 01 05 (31.3)
31‑40 27 14 41 (36.6) 05 03 08 (50)
41‑50 13 06 19 (17) 02 0 02 (12.5)
51‑60 04 02 06 (5.3) 01 0 01 (6.2)
≥61 02 0 02 (1.8) 0 0 0
Total 77 (68.8) 35 (31.2) 112 (100) 12 (75) 04 (25) 16 (100)
CM: Cryptococcal meningitis, PLHAs: People living with HIV/AIDS
325 Journal of Natural Science, Biology and Medicine | July 2014 | Vol 5 | Issue 2
Dash, et al.: Cryptococcal meningitis among PLHAs
C. neoformans, whereas both microscopy and fungal culture
identified all 16 positive cases. The CD4 T‑lymphocytes
count of all these cases except 1 case were <100 cells/µl
All laboratories confirmed CM patients were given
induction therapy with amphotericin B and fluconazole
daily for 14 days during their hospitalization. After clinical
improvement, patients were discharged with the advice of
taking tablet fluconazole 400 mg daily for 8 weeks followed
by fluconazole 200 mg daily for 12 months. Unfortunately,
2 patients were expired during their hospital stay. The mean
duration of hospital stay was 10 days (range from 7 days
to 16 days).
The asexual yeast C. neoformans has been classified as
four serotypes based on the capsular polysaccharide,
glucurononoxylomannan. Capsular types A through D
correspond to the variants C. neoformans var. grubii (A),
C. neoformans var. gattii (B and C), and C. neoformans var.
neoformans (D). Recently, Cryptococcus gattii has been classified
as a separate species as it has shown to be genetically distinct
from C. neoformans. It is a well‑recognized opportunistic
infection among cell‑mediated immunodeficient patients,
such as HIV infection, organ transplantation, and
rheumatologic conditions requiring immunosuppressive
agents. In HIV patients, it is classified as an acquired
immunodeficiency syndrome (AIDS)‑defining condition.
Characteristics of Cryptococcus that permit its survival within
the host include a polysaccharide capsule, and phenol
oxidase enzyme uses catecholamine as a substrate to
produce melanin, which accumulates in the cell wall. It is
the use of catecholamine that may produce a predilection
for involvement of the CNS.
Typically, CNS opportunistic infection occurs during
severe immune deficiency in advanced HIV infection when
CD4 T‑lymphocytes count is less than 200 cells/µl.
Cryptococcus meningitis caused by C. neoformans is one of the
most common opportunistic CNS infections in PLHAs.
Before the introduction of ART, 5‑10% of patients with
AIDS developed CM. Although the incidence has fallen,
this disease remains a major concern in sub‑Saharan
Africa and south and southeast Asia. In our study, the
prevalence observed was 14.3% (16 out of 112). This
value is comparable with the reports prepared by Lakshmi
et al., they found out 10.86% (39 out of 359) of suspected
CM cases showed a definite diagnosis of C. neoformans.
Various studies have been conducted in different parts
of the world including India, to find the prevalence of
CM in HIV‑seropositive patients and has been found to
vary widely from 2.79% to 55%.[13,20‑22] This discrepancy
probably is due to under reporting and misdiagnosis of
Three successive studies conducted in AIIMS, New Delhi
over a period of 12 years (1992‑2004) had revealed
that parallel to increase in number of HIV cases; HIV
cryptococcosis co‑infection increased from 20% in 1992‑96
to 30% in 1996‑2000 and 49% in 2000‑04. In this
present study no such increase in prevalence over 2½ years
was observed (P is 1.000, not significant). Other studies
conducted in India did not find any significant increase in
the prevalence of CM over the years.[12,22]
Our study revealed, male in the age group of 21‑40 years
were most commonly affected than female, which may
reflect a difference of exposure and out‑door activity
rather than a difference in host susceptibility as it was noted
earlier. The present study did not find any confirmed CM
cases in the age group of 0‑20 years. Though children are
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Figure 1: Clinical presentations of laboratory confirmed cases of
cryptococcal meningitis in people living with human immunodeficiency
virus/acquired immunodeficiency syndrome
Journal of Natural Science, Biology and Medicine | July 2014 | Vol 5 | Issue 2 326
Figure 2: CD4 T‑lymphocytes count of laboratory confirmed
cryptococcal meningitis cases
Dash, et al.: Cryptococcal meningitis among PLHAs
less commonly affected, now there is an increase in the
prevalence of CM observed in HIV‑infected children.[24,25]
The clinical manifestations observed in the laboratory
confirmed cases were fever and headache (100%), followed
by altered sensorium (93.7%), neck stiffness (75%),
vomiting (62.5%), and wasting (56.2%). Baradkar et al.
noted headache, fever and altered sensorium (100%),
neck stiffness (90%) and vomiting was present in 52.6%
cases, similar to our study. Our findings are also
comparable with a study conducted by Lakshmi et al. in
India. Present study differed from Lee et al., they reported
fever (72.7%), headache (54.5%), altered mentality (45.5%),
dyspnea (36.4%), general weakness (27.3%), dizziness,
insomnia, and vomiting (18.2%) cases in South Korea.
Other presentations such as lethargy, coma, papilledema,
focal neurological deficits, and cranial neuropathies
were observed by different workers, no such similar
complications were noted in our study.
Negative staining with 10% Nigrosin revealed 87.5%
confirmed cases of C. neoformans, but the combination of
negative staining (10% Nigrosin) and fungal culture had
detected all 16 cases of C. neoformans, in this present study.
Microscopic detection with India ink or 10% Nigrosin and
fungal culture of CSF are diagnostic for CM. This present
study showed in 93.7% of laboratory confirmed cases
CD4 T‑lymphocytes count was 100 cells/µl and only in
1 case, the CD4 T‑lymphocytes count was 137 cells/µl.
Lakshmi et al. reported in all confirmed CM cases the CD4
T‑lymphocytes count was 100/µl. In our study, no case
of concomitant cryptococcal and tubercular meningitis
was detected, similar to study conducted by Thakur et al.
In comparison, Lakshmi et al. had reported concomitant
meningitis up to 33% of AIDS patients in India.
All 16 patients in our study responded initially to induction
therapy of IV amphotericin B 1 mg/kg and fluconazole
800 mg daily for 2 weeks. During their discharge, patient
were advised to take oral fluconazole 400 mg daily
for 8 weeks followed by fluconazole 200 mg daily for
12 months and frequent follow‑up. Unfortunately, 4 (25%)
patients were lost for follow‑up and 2 (12.5%) patients
expired during their hospital stay. No relapse was observed
among recovered patients.
CM remains a significant cause of morbidity and mortality
particularly among PLHAs in resource poor environments.
Present study indicates a high prevalence of CM in
PLHAs in a tertiary care hospital setting. As the clinical
and radiological pictures of CM are often non‑pointing,
routine mycological evaluation is necessary for early definite
diagnosis and subsequent initiation of appropriate therapy
as the majority of patients respond well to treatment if
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How to cite this article: Dash M, Padhi S, Sahu R, Turuk J, Patta-naik
S, Misra P. Prevalence of cryptococcal meningitis among people
living with human immunodeficiency virus/acquired immunodeficiency
syndrome in a Tertiary Care Hospital, Southern Odisha, India. J Nat Sc
Biol Med 2014;5:324-8.
Source of Support: Nil. Conflict of Interest: None declared.
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