Cryptococcal meningitis in HIV-infected patients: a longitudinal study in Cambodia.

Abstract

To describe the frequency of diagnosis of cryptococcosis among HIV-infected patients in Phnom Penh, Cambodia, at programme entry, to investigate associated risk factors, and to determine the incidence of cryptococcal meningitis.
We analysed individual monitoring data from 11,970 HIV-infected adults enrolled between 1999 and 2008. We used Kaplan-Meier naïve methods to estimate survival and retention in care and multiple logistic regression to investigate associations with individual-level factors.
Cryptococcal meningitis was diagnosed in 12.0% of the patients: 1066 at inclusion and 374 during follow-up. Incidence was 20.3 per 1000 person-years and decreased over time. At diagnosis, median age was 33 years, median CD4 cell count was 8 cells/μl, and 2.4% of patients were receiving combined antiretroviral therapy; 38.7% died and 34.6% were lost to follow-up. Of 750 patients alive and in care after 3 months of diagnosis, 85.9% received secondary cryptococcal meningitis prophylaxis and 13.7% relapsed in median 5.7 months [interquartile range 4.1-8.8] after cryptococcal meningitis diagnosis (relapse incidence=5.7 per 100 person-years; 95%CI 4.7-6.9). Cryptococcal meningitis was more common in men at programme entry (adjusted OR=2.24, 95% CI 1.67-3.00) and fell with higher levels of CD4 cell counts (P<0.0001).
Cryptococcal meningitis remains an important cause of morbidity and mortality in Cambodian HIV-infected patients. Our findings highlight the importance of increasing early access to HIV care and cryptococcal meningitis prophylaxis and of improving its diagnosis in resource-limited settings.

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Cryptococcal meningitis in HIV-infected patients: a longitudinal
study in Cambodia
Emmanuelle Espie ´1, Loretxu Pinoges1, Suna Balkan2, Ngeth Chanchhaya3, Lucas Molfino4, Prak Narom3and
Mar Pujades-Rodrı ´guez1
1 Epicentre, Paris, France
2 Me ´decins Sans Frontie `res-France, Paris, France
3 Khmer-Soviet Friendship Hospital, Phnom Penh, Cambodia
4 Me ´decins Sans Frontie `res-France, Phnom Penh, Cambodia
Summary
objective To describe the frequency of diagnosis of cryptococcosis among HIV-infected patients in
Phnom Penh, Cambodia, at programme entry, to investigate associated risk factors, and to determine the
incidence of cryptococcal meningitis.
methods We analysed individual monitoring data from 11 970 HIV-infected adults enrolled between
1999 and 2008. We used Kaplan–Meier naı ¨ve methods to estimate survival and retention in care and
multiple logistic regression to investigate associations with individual-level factors.
results Cryptococcal meningitis was diagnosed in 12.0% of the patients: 1066 at inclusion and 374
during follow-up. Incidence was 20.3 per 1000 person-years and decreased over time. At diagnosis,
median age was 33 years, median CD4 cell count was 8 cells⁄ll, and 2.4% of patients were receiving
combined antiretroviral therapy; 38.7% died and 34.6% were lost to follow-up. Of 750 patients alive
and in care after 3 months of diagnosis, 85.9% received secondary cryptococcal meningitis prophylaxis
and 13.7% relapsed in median 5.7 months [interquartile range 4.1–8.8] after cryptococcal meningitis
diagnosis (relapse incidence = 5.7 per 100 person-years; 95%CI 4.7–6.9). Cryptococcal meningitis was
more common in men at programme entry (adjusted OR = 2.24, 95% CI 1.67–3.00) and fell with higher
levels of CD4 cell counts (P < 0.0001).
conclusions Cryptococcal meningitis remains an important cause of morbidity and mortality in
Cambodian HIV-infected patients. Our findings highlight the importance of increasing early access to
HIV care and cryptococcal meningitis prophylaxis and of improving its diagnosis in resource-limited
settings.
keywords Asia, cryptococcosis, opportunistic infection, HIV, epidemiology
Introduction
Cryptococcus neoformans is an encapsulated fungal
pathogen that causes cryptococcosis, a life-threatening
condition common in patients with defective cell-
mediated immunity. It is the third most frequent
opportunistic infection of the central nervous system in
HIV-infected individuals (Powderly 1993). In the pre-
antiretroviral therapy area, cryptococcosis was a lead-
ing cause of mortality in patients hospitalised with
AIDS in Cambodia (Senya et al. 2003; Chhin et al.
2004; Kong et al. 2007; Madec et al. 2007). Reported
prevalence among patients with AIDS varies from 2 to
10% in Europe (Dromer et al. 2004; Antinori et al.
2009) and North America (Sorvillo et al. 1997; Mirza
et al. 2003), but it is more than 15% in Africa (Moosa
& Coovadia 1997; Heyderman et al. 1998; French et al.
2002; Jarvis et al. 2009) and more than 20% in Asia
(Thailand) (Chariyalertsak et al. 2001; Amornkul et al.
2003).
In this study, we describe the frequency of cryptococcal
meningitis (CM) in a large cohort of HIV-infected
individuals treated in the Me ´decins Sans Frontie `res
(MSF)-supported HIV⁄AIDS programme in Phnom Penh,
Cambodia at programme entry; and incidence of disease,
CM-associated mortality, relapse rates and individual-level
factors associated with cryptococcosis diagnosis at
programme entry.
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Methods
All HIV-infected adults (‡15 years old) who received free
medical care in the MSF-supported HIV⁄AIDS programme
in Phnom Penh between February 1999 and November
2008 were eligible for inclusion. Hospital and outpatient
care is offered at the Khmer-Soviet Friendship Hospital, the
national referral centre for HIV in Cambodia. Free com-
bined antiretroviral therapy (ART) provision was started in
June 2001 and primary prophylaxis against CM in 2002.
Medical follow-up visits for patients on ART are
scheduled every 3 months after clinical stabilisation.
Primary prophylaxis with fluconazole (200 mg⁄day) is
given to patients with CD4 cell counts <50 cells⁄ll, and
<100 cells⁄ll since 2004. Diagnosis of CM is suspected in
patients with severe headache, fever, neck stiffness, altered
mental status, neurological deficit and⁄or coma. Confir-
mation is made with India ink direct examination of the
cerebrospinal fluid. Since 2006, Pastorex Crypto Plus (Bio-
Rad Laboratories, Hercules, CA, USA) has been used to
detect serum cryptococcal antigen for patients with CD4
cell count below 100 cells⁄ll.
Patients diagnosed with CM receive amphotericin B
(0.7 mg⁄kg⁄day for 2 weeks), followed by fluconazole
(400 mg⁄day for 8 weeks). Culture for C. neoformans is
performed in patients with persistent symptoms after
receiving 14 days of antifungal treatment to confirm
clearance of the pathogen. Secondary prophylaxis with
fluconazole (200 mg⁄day) is routinely provided after
treatment completion and until CD4 cell counts exceed
100 cells⁄ll.
We analysed routine individual patient data collected
using standardised forms and entered into the FUCHIA
software (Epicentre, Paris, France). Data included birth
date, sex, weight, height, new or recurrent opportunistic
infections (e.g. CM, penicilliosis, tuberculosis or histo-
plasmosis), primary and secondary antifungal prophylaxis
(e.g. fluconazole and itraconazole), ART regimen and
starting date, CD4 lymphocyte counts with date of blood
collection, and dates of visit, hospitalisation, death or
transfer to another programme.
We categorised body mass index (BMI; <16, 16–18.4
and ‡18.5 kg⁄m2), age (15–24, 25–39 and ‡40 years) and
time period at programme inclusion (pre-ART, 2001–
2002, 2003–2005, and 2006–2008). Measurements of
CD4 cell count closest to and between 3 months before
and up to 1 month after programme inclusion, and closest
to and within 2 months of CM diagnosis, were grouped
into <20, 20–49, 50–99, ‡100 cells⁄ll, and missing.
At cohort inclusion, we defined cases of CM as those
diagnosed within 15 days of enrolment, incident cases as
first recorded episodes diagnosed thereafter, and relapses as
new episodes of CM diagnosed three or more months after
the first recorded episode.
Statistical analysis
Patient follow-up was right-censored at the earliest of the
following dates: CM diagnosis (or relapse), death, transfer
out or last visit. Overall incidence of first recorded episode
of CM and of first relapse were then estimated with
Poisson exact 95% confidence intervals. Incidence was also
described by age group, sex, and calendar period at
programme inclusion and was calculated in the subgroup
of patients who had not been diagnosed with CM at
programme entry. Relapse rates were estimated in the
subgroup of patients with ‡3 months of follow-up after
CM diagnosis. Kaplan–Meier methods were used to
estimate probabilities of incident CM by CD4 count level
at programme inclusion.
Probabilities of survival among patients who were not
lost to follow-up and of retention in care were calculated
using Kaplan–Meier naı ¨ve methods. These were then
compared to patients with or without cryptococcal
diagnosis using log-rank tests. Patients who missed their
last scheduled appointment for ‡2 months were consid-
ered lost to follow-up. Finally, we identified factors
associated with CM diagnosis at programme entry using
multiple logistic regression and log-likelihood ratio tests
for association and trend. A priori confounders were sex,
age group and year of programme inclusion and models
were further adjusted for factors associated with the
outcome in univariable analyses (P < 0.2). Statistical
analyses were performed using Stata 10.1 (Stata Corp.,
College Station, TX, USA). All significance tests were
two-sided, and P values of 0.05 or less were considered
significant.
Results
During the study period, 11 970 HIV-infected adults
entered the programme and contributed 18 409 person-
years to the analysis. Twenty-four patients (0.2%) with
missing age, one diagnosed with CM, were excluded. At
cohort inclusion, median age was 33 years [interquartile
range (IQR) 29–38] and 42.8% of patients were women.
Median duration of follow-up in the programme was
6.2 months [IQR 0.8–32.4] and 4339 patients initiated
ART in median 6.9 months after programme inclusion
[IQR 2.5–14.1]. At ART start, median CD4 cell count was
43 cells⁄ll [IQR 10–130; n = 3631], and 77.5% of the
2471 patients who were eligible for primary prophylaxis
against cryptococcosis received it. CM was diagnosed in
1440 patients (12.0% of the cohort).
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Cryptococcal meningitis diagnosis at programme entry
and incidence
At cohort inclusion, 1066 patients (8.9% of patients) were
diagnosed with CM (Table 1). Frequency of cryptococcal
diagnosis remained stable over time and, in all age groups,
was higher in men than in women. The highest frequency
was observed in the 25–39 year group.
A total of 374 individuals were diagnosed with CM
during follow-up. This represents an incidence rate of 20.3
per 1000 person-years (95% CI 18.4–22.5). The highest
incidence was observed in the pre-ART era (1999–2000)
with 36.8 per 1000 person-years, which decreased gradu-
ally to 11.8 per 1000 person-years in the 2006–2008
period. As with prevalence, incidence was higher in men
than in women for all age groups, and higher rates were
observed in patients aged 25 years or older. At the time of
diagnosis, 45 (12.0%) individuals were receiving primary
antifungal prophylaxis for a median of 66 days [IQR
29–136]. Cumulative probability of incident CM
increased with lower CD4 count levels at programme entry
(Figure 1).
Characteristics of patients at cryptococcal meningitis
diagnosis
Of the 1440 patients ever diagnosed with CM, 370
(25.7%) were women. At the time of cryptococcosis
diagnosis, median age was 33 years [IQR 29–38], and 34
(2.4%) patients were receiving ART for a median of
35 days [IQR 20–92] (Table 2).
Median CD4 cell count at cryptococcosis diagnosis was
17 cells⁄ll [IQR 7–34; n = 189], and 80.4% of patients
had counts less than 50 cells⁄ll. Men and patients with
lower CD4 count levels at programme inclusion were more
likely to be diagnosed with CM than women (adjusted
odds ratio 2.2, 95%CI 1.7–3.0; P < 0.0001), and patients
with higher CD4 cell counts (P for trend <0.001; Table 3).
Table 1 Cryptococcal meningitis at programme inclusion, incidence of cryptococcal meningitis and cryptococcal relapse by calendar
period of enrolment, sex and age group, Phnom Penh, Cambodia, 1999–2008
Cryptococcal meningitis at
programme entry
Cryptococcal meningitis during
follow-up* Cryptococcal meningitis relapse
Number
of cases
Percentage of
patients (95% CI)
Number
of cases
Incidence per
1000 PY (95% CI)
Number
of cases
Incidence per
100 PY (95% CI)
Total
Calendar period
1999–2000
2001–2002
2003–2005
2006–2008
Sex
Women
Men
Age group, years
15–24
25–39
‡40
1066 8.9 (8.4–9.4)374 20.3 (18.4–22.5)103 5.7 (4.7–6.9)
182
316
419
149
9.9 (6.6–9.0)
8.9 (8.1–9.8)
9.0 (7.9–9.8)
7.8 (8.6–11.3)
58
157
137
22
36.8 (28.4–47.6)
23.6 (20.2–27.6)
16.5 (14.0–19.5)
11.8 (7.8–17.9)
29
40
27
7
31.9 (22.2–45.9)
5.4 (3.9–7.3)
3.2 (2.2–4.7)
5.1 (2.5–10.8)
264
802
5.1 (4.5–5.7)
11.7 (10.9–12.5)
106
268
10.7 (8.9–13.0)
31.5 (27.9–35.5)
17
86
4.4 (2.8–7.1)
6.0 (4.9–7.4)
62 5.7 (4.3–7.1)
9.7 (9.0–10.3)
7.8 (6.8–8.8)
26
272
76
13.9 (9.5–20.4)
21.4 (19.0–24.1)
19.7 (15.8–24.7)
5 6.0 (2.5–14.4)
6.0 (4.9–7.5)
3.9 (2.2–6.8)
799
205
86
12
95%CI, 95% binomial exact confidence interval; PY, person-years of follow-up.
*Only first diagnosed incident episode of cryptococcal meningitis considered among patients with no cryptococcal meningitis at
programme entry.
0.00
0.02
0.04
0.06
0.08
0.10
Cumulative probability
0369 12
Time since program inclusion (months)
<20 cells/µl
50–99 cells/µl
20–49 cells/µl
100 + cells/µl
Figure 1 Cumulative probability of incident cryptococcal men-
ingitis per CD4 cell count level at programme inclusion, Phnom
Penh, Cambodia, 1999–2008.
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Calendar year, age, or BMI at inclusion were not associ-
ated with diagnosis of CM at programme inclusion.
Almost all patients diagnosed with CM (96.5%)
required hospitalisation for a median duration of 21 days
[IQR 14–27]. Concomitant opportunistic infections diag-
nosed were pulmonary (n = 188; 13.1%) and extrapul-
monary tuberculosis (n = 218; 15.1%).
Treatment outcomes, relapse rates, and secondary
prophylaxis
By November 2008, 338 (23.5%) of patients diagnosed
with CM were alive and followed in the cohort; 47 had
been transferred to another HIV programme in median
37 months after diagnosis; 498 (34.6%) had been lost to
follow-up after a median time of 52 days [IQR 21–151];
and 557 (38.7%) had died after 26 days [IQR 6–154].
Programme retention was lower and mortality higher in
patients diagnosed with CM than in other patients (log-
rank test P < 0.0001; Figures 2a,b). For patients diagnosed
with CM, probabilities of retention in care at 6, 12, 24, and
36 months of enrolment were 0.50, 0.37, 0.29, and 0.27,
respectively.
Among the 750 patients alive and in care after 3 months
of CM diagnosis, 644 (85.9%) received secondary pro-
phylaxis for cryptococcosis and 151 (20.1%) received
ART. A total of 103 patients (13.7%) relapsed in median
5.7 months [IQR 4.1–8.8] after cryptococcosis diagnosis.
This represented a relapse rate of 5.7 per 100 person-years
(95% CI 4.7–6.9). Patients who experienced relapse had
median CD4 cell counts of 6 cells⁄ll at ART initiation
[IQR 2–14; n = 53] and 14.5 cells⁄ll [IQR 6–34.5; n = 28]
at first recorded cryptococcosis diagnosis. Seventy-one
(69%) patients were receiving secondary cryptococcosis
prophylaxis at the time of relapse, and 36 (35%) had
started ART in median 3.8 months before the time of
relapse [IQR 1.7–7.4]. Forty-three (41.7%) patients died in
median 3.2 months [IQR 0.6–7.1] after relapse diagnosis,
and 25 (24.3%) were lost to follow-up after 3.0 months
[IQR 1.0–4.9].
Discussion
In this observational open cohort followed between 1999
and 2008 in Cambodia, 9% of HIV-infected patients were
diagnosed with CM at programme inclusion; incidence
was 20.3 per 1000 person-years and decreased over time
from 36.8 per 1000 person-years in the pre-ART period
to 11.8 per 1000 person-years in 2006–2008, and relapse
rate was 5.7 per 100 person-years.
In our setting, CM remained a common opportunistic
infection among HIV-infected patients at programme
inclusion. Even if the proportion of patients diagnosed with
CM at programme entry remained stable over time, the
Table 2 Characteristics of HIV-infected
patients with cryptococcal meningitis at
diagnosis of disease, Phnom Penh, Cambo-
dia, 1999–2008
Cryptococcal meningitis
Total
(N = 1440)
Cases diagnosed at
programme entry
(N = 1066)
Incident cases
(N = 374)
Median age, years [IQR]
Median BMI, kg⁄m2[IQR]
33 [29–38]
n = 489
17.9 [15.8–19.8]
n = 458
8 [3–23]
327 (71.4)
76 (16.6)
32 (7.0)
23 (5.0)
34 (2.4)
33 [29–38]
n = 318
18.0 [16.0–20.1]
n = 306
8.5 [3–22]
218 (71.2)
49 (16.0)
27 (8.8)
12 (3.9)
–
33 [29–38]
n = 171
17.7 [15.4–19.5]
n = 152
7 [3–25]
109 (71.7)
27 (17.8)
5 (3.3)
11 (7.2)
34 (9.1)
CD4 cell count, cells⁄ll
Median [IQR]
<20 (%)
20–49 (%)
50–99 (%)
‡100 (%)
Receiving ART
at diagnosis (%)
Hospitalised during
episode (%)
Median time of follow-up,
months* [IQR]
Number of deaths (%)
Median time to death*,
days [IQR]
1389 (96.5) 1,038 (97.4)351 (93.8)
3.4 [0.6–21.6]3.0 [0.5–15.6] 5.2 [1.0–46.7]
557 (38.7)
26 [6–154]
418 (39.2)
19 [4–147]
139 (37.2)
45 [14–188]
ART, combined antiretroviral therapy; BMI, body mass index; IQR, interquartile range.
*Time since cryptococcosis diagnosis.
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absolute number of cases diagnosed doubled between
1999–2000 and 2003–2005. Incidence was highest in the
earlier calendar period and decreased over time to 11.8 per
1000 person-years in 2006–2008. This decrease might
partly be explained by the observed gradual increase in
patient CD4 count levels at programme entry, which in
turn would lead to reduce the length of time when patients
are at increased risk of developing cryptococcosis
(<50 cells⁄ll). High proportions of patients diagnosed with
CM at presentation have also been reported in previous
studies conducted in Cambodia (11.8% to 18%) (Pichith
et al. 2001; Senya et al. 2003; Chhin et al. 2004; Kong
et al. 2007; Micol et al. 2007); Thailand and Vietnam
(18% to 36% of patients with AIDS) (Chariyalertsak et al.
2001; Amornkul et al. 2003; Louie et al. 2004; Jongwuti-
wes et al. 2007; Manosuthi et al. 2007). The observed high
frequency of diagnosis at programme inclusion is the result
of the late presentation of HIV patients for treatment.
Median CD4 cell count at programme inclusion was below
50 cells⁄ll and, as in other countries (Darras-Joly et al.
1996; Sungkanuparph et al. 2005; Antinori et al. 2009),
most patients with CM arrived with severe neurological
symptoms (e.g. coma, confusion) and were diagnosed with
both HIV and cryptococcosis at hospitalisation. As previ-
ously reported, we found that men were twice as likely as
women to be diagnosed with CM at programme inclusion
(Sorvillo et al. 1997; Amornkul et al. 2003; Dromer et al.
2004; Micol et al. 2007), which might relate to differences
in either level of exposure or susceptibility to the pathogen.
Table 3 Associations between characteristics at programme
inclusion and cryptococcal meningitis diagnosed at programme
entry, Phnom Penh, Cambodia, 1999–2008
Adjusted OR95% CIP-value
Sex
Women
Men
CD4 cell count, cells⁄ll
<20
20–49
50–99
‡100
Calendar year
1999–2000
2001–2002
2003–2005
2006–2008
Age, years
15–24
25–39
‡40
BMI, kg⁄m2
‡18.5
16–18.4
<16
Ref
2.24
–
1.67–3.00<0.0001
Ref
0.54
0.28
0.07
–
0.38–0.75
0.18–0.43
0.03–0.12
<0.0001
Ref
1.02
1.10
0.87
–
0.51–2.09
0.55–2.20
0.42–1.78
0.54
Ref
1.80
1.48
–
0.92–3.50
0.72–3.00
0.10
Ref
0.93
1.29
–
0.69–1.24
0.92–1.81
0.17
95% CI, 95% confidence interval; BMI, body mass index;
OR, odds ratio from multiple logistic regression adjusted for age,
BMI, CD4 cell count, year of inclusion and sex; P-value from
likelihood ratio test for association.
Mortality among patients not lost to follow-up
1.00
0.0
0.2
0.4
0.6
0.8
Cumulative probability
012 24 366 1830
Time since program inclusion (months)
No cryptococcosis
Number at
risk
No CM4021 36283242 2859
2607 2401
CM 540441 384342 314297
Program attrition among all patients
0.0
0.4
0.2
0.6
0.8
1.0
Cumulative probability
012 24 366
Time since program inclusion (months)
18 30
No cryptococcosis
Number
at risk
No CM 5365430136623129 2789 2531
CM 685 497 416 363328 305
(a)
(b)
Cryptococcosis
Cryptococcosis
Figure 2 Cumulative mortality and attrition after programme
inclusion in patients diagnosed or not with cryptococcal menin-
gitis, Phnom Penh, Cambodia, 1999–2008. (a) Mortality among
patients not lost to follow-up. (b)Programme attrition among all
patients.
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Cryptococcosis-related deaths represented 25% of all
recorded deaths in our cohort, and case fatality rate among
patients diagnosed with CM was 39%, confirming that
CM is a leading cause of early mortality among patients
with HIV⁄AIDS in Phnom Penh. Besides promoting earlier
HIV testing, diagnosis, and treatment for HIV-infected
patients to prevent late presentation of CM disease, patient
education on signs and symptoms of severe illness should
routinely be provided. Systematic testing of serum crypto-
coccal polysaccharide has been shown to be clinically
valuable for detection of subclinical cryptococcosis in
patients with CD4 counts <100 cells⁄ll (Micol et al.
2007), but this test is technically complicated to perform
and costly for routine use in resource-limited countries. It is
therefore also important that adequate diagnostic tools for
CM are made available in resource-limited settings.
Other contributing factors to high mortality in our
setting are likely to be related to the difficulties in
managing severe cases at hospital level, including imple-
mentation of intravenous amphotericin B treatment in
settings where renal function cannot easily be monitored.
Moreover, in our cohort, 66% of the patients who were
diagnosed with CM at programme inclusion and survived
2 months after diagnosis had not been started on ART.
Starting ART as soon as the condition of the patient allows
should be encouraged to increase patient survival.
Primary chemoprophylaxis with fluconazole is recom-
mended for patients with CD4 cell counts <100 cells⁄ll
(Singh et al. 1996; Havlir et al. 1998; Micol et al. 2007).
In our cohort, 78% of eligible patients actually received it
and only 6% of these developed CM. This finding stresses
the importance of a prompt start of primary prophylaxis
against cryptococcosis.
The relapse rate observed in our setting was similar to
that reported in a multicentre cohort of patients in France
(4.6 per 100 person-years) (Lortholary et al. 2006), but
episodes were diagnosed earlier during follow-up (median
of 6 compared with 12 months in the French cohort).
Earlier relapse episodes could be explained by late start of
ART, unavailability of flucytosine for cryptococcosis
treatment, and⁄or inadequate adherence to antifungal
therapy or secondary prophylaxis.
In this analysis of a large cohort of HIV-infected
patients, we analysed routinely collected individual patient
data. We lacked detailed information on case severity (e.g.
presence of coma or impossibility to eat), results of
cerebrospinal fluid examinations (detection and quantifi-
cation of C. neoformans antigen), or information related to
prescribed antifungal therapy. However, the availability of
good-quality data collected over an 8-year period and the
large sample size achieved allowed describing trends in
both CM diagnosis at programme entry and incidence over
time and obtaining precise estimations in the risk factor
analysis.
Conclusions
Our findings indicate that efforts still need to be made to
increase early diagnosis and treatment of HIV infection
and disease, provide primary and secondary prophylaxis
against cryptococcosis, and early initiate ART for patients
with CM. In parallel, early diagnosis of CM and
improvement in acute management of the disease are
needed. New strategies are therefore called for to reinforce
and maintain treatment adherence and prophylaxis to
decrease cryptococcal-related case fatality and relapse in
resource-limited countries.
Acknowledgements
We thank the personnel of the Ministry of Health working
in the Infectious Disease Department of the Khmer-Soviet
Friendship Hospital of Phnom Penh for their daily efforts
and dedicated work. We are also grateful to the field team
of Me ´decins Sans Frontie `res who ensure regular collection,
entry and cleaning of clinical patient data; and to the
Epicentre FUCHIA team for their work maintaining and
improving the quality of the data used in this analysis. We
finally thank Jean-Franc ¸ois Etard for his advice on this
manuscript and to Oliver Yun for his editorial work. The
work was funded by Me ´decins Sans Frontie `res – France.
References
Amornkul PN, Hu DJ, Tansuphasawadikul S et al. (2003) Human
immunodeficiency virus type 1 subtype and other factors asso-
ciated with extrapulmonary Cryptococcosis among patients in
Thailand with AIDS. AIDS Research of Human Retroviruses
19, 85–90.
Antinori S, Ridolfo A, Fasan M et al. (2009) AIDS-associated
cryptococcosis: a comparison of epidemiology, clinical features
and outcome in the pre- and post-HAART eras. Experience of a
single centre in Italy. HIV Medicine 10, 6–11.
Chariyalertsak S, Sirisanthana T, Saengwonloey O & Nelson KE
(2001) Clinical presentation and risk behaviors of patients with
acquired immunodeficiency syndrome in Thailand, 1994–1998:
regional variation and temporal trends. Clinical Infectious
Diseases 32, 955–962.
Chhin S, Rozycki G, Pugatch D & Harwell JI (2004) Aetiology of
meningitis in HIV-infected patients in a referral hospital in
Phnom Penh, Cambodia. International Journal of STD & AIDS
15, 48–50.
Darras-Joly C, Chevret S, Wolff M et al. (1996) Cryptococcus
neoformans infection in France: epidemiologic features of and
early prognostic parameters for 76 patients who were infected
Tropical Medicine and International Healthvolume 15 no 11 pp 1375–1381 november 2010
E. Espie ´ et al. Cryptococcal meningitis in HIV-infected patients
1380
ª 2010 Blackwell Publishing Ltd

Page 7

with human immunodeficiency virus. Clinical Infectious
Diseases 23, 369–376.
Dromer F, Mathoulin-Pelissier S, Fontanet A, Ronin O, Dupont B
& Lortholary O (2004) Epidemiology of HIV-associated cryp-
tococcosis in France (1985-2001): comparison of the pre- and
post-HAART eras. AIDS 18, 555–562.
French N, Gray K, Watera C et al. (2002) Cryptococcal infection
in a cohort of HIV-1-infected Ugandan adults. AIDS 16, 1031–
1038.
Havlir DV, Dube MP, McCutchan JA et al. (1998) Prophylaxis
with weekly versus daily fluconazole for fungal infections in
patients with AIDS. Clinical Infectious Diseases 27, 1369–1375.
Heyderman RS, Gangaidzo IT, Hakim JG et al. (1998) Crypto-
coccal meningitis in human immunodeficiency virus-infected
patients in Harare, Zimbabwe. Clinical Infectious Diseases 26,
284–289.
Jarvis JN, Boulle A, Loyse A et al. (2009) High ongoing burden of
cryptococcal disease in Africa despite antiretroviral roll out.
AIDS 23, 1182–1183.
Jongwutiwes U, Kiertiburanakul S & Sungkanuparph S (2007)
Impact of antiretroviral therapy on the relapse of cryptococcosis
and survival of HIV-infected patients with cryptococcal infec-
tion. Current HIV Research 5, 355–360.
Kong BN, Harwell JI, Suos P et al. (2007) Opportunistic infections
and HIV clinical disease stage among patients presenting for
care in Phnom Penh, Cambodia. Southeast Asian Journal of
Tropical Medicine and Public Health 38, 62–68.
Lortholary O, Poizat G, Zeller V et al. (2006) Long-term outcome
of AIDS-associated cryptococcosis in the era of combination
antiretroviral therapy. AIDS 20, 2183–2191.
Louie JK, Chi NH, Thao LT et al. (2004) Opportunistic infections
in hospitalized HIV-infected adults in Ho Chi Minh City,
Vietnam: a cross-sectional study. International Journal of STD
& AIDS 15, 758–761.
Madec Y, Laureillard D, Pinoges L et al. (2007) Response to
highly active antiretroviral therapy among severely immuno-
compromised HIV-infected patients in Cambodia. AIDS 21,
351–359.
Manosuthi W, Chaovavanich A, Tansuphaswadikul S et al. (2007)
Incidence and risk factors of major opportunistic infections after
initiation of antiretroviral therapy among advanced HIV-
infected patients in a resource-limited setting. Journal of
Infection 55, 464–469.
Micol R, Lortholary O, Sar B et al. (2007) Prevalence, determi-
nants of positivity, and clinical utility of cryptococcal antigen-
emia in Cambodian HIV-infected patients. Journal of Acquired
Immune Deficiency Syndromes 45, 555–559.
Mirza SA, Phelan M, Rimland D et al. (2003) The changing epi-
demiology of cryptococcosis: an update from population-based
active surveillance in 2 large metropolitan areas, 1992-2000.
Clinical Infectious Diseases 36, 789–794.
Moosa MY & Coovadia YM (1997) Cryptococcal meningitis in
Durban, South Africa: a comparison of clinical features, labo-
ratory findings, and outcome for human immunodeficiency virus
(HIV)-positive and HIV-negative patients. Clinical Infectious
Diseases 24, 131–134.
Pichith K, Chanroeun H, Bunna P et al. (2001) Clinical aspects
of AIDS at the Calmette hospital in Phnom Penh, Kingdom
of Cambodia A report on 356 patients hospitalized in the
Medicine B Department of the Calmette Hospital. Sante ´ 11,
17–23.
Powderly WG (1993) Cryptococcal meningitis and AIDS. Clinical
Infectious Diseases 17, 837–842.
Senya C, Mehta A, Harwell JI, Pugatch D, Flanigan T & Mayer
KH (2003) Spectrum of opportunistic infections in hospitalized
HIV-infected patients in Phnom Penh, Cambodia. International
Journal of STD & AIDS 14, 411–416.
Singh N, Barnish MJ, Berman S et al. (1996) Low-dose fluconazole
as primary prophylaxis for cryptococcal infection in AIDS
patients with CD4 cell counts of < or = 100⁄mm3: demonstra-
tion of efficacy in a positive, multicenter trial. Clinical Infectious
Diseases 23, 1282–1286.
Sorvillo F, Beall G, Turner PA, Beer VL, Kovacs AA & Kerndt PR
(1997) Incidence and factors associated with extrapulmonary
cryptococcosis among persons with HIV infection in Los
Angeles County. AIDS 11, 673–679.
Sungkanuparph S, Kiertiburanakul S, Manosuthi W,
Kiatatchasai W & Vibhagool A (2005) Initiation of highly
active antiretroviral therapy in advanced AIDS with CD4 < 50
cells⁄mm3 in a resource-limited setting: efficacy and
tolerability. International Journal of STD & AIDS 16,
243–246.
Corresponding Author Mar Pujades-Rodrı ´guez, Epicentre, Me ´decins Sans Frontie `res, 8, rue Saint Sabin, 75011 Paris, France.
Tel.: +33 4 72115736; Fax: +33 4 72115141; E-mail: mar.pujades@epicentre.msf.org
Tropical Medicine and International Health volume 15 no 11 pp 1375–1381 november 2010
E. Espie ´ et al. Cryptococcal meningitis in HIV-infected patients
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