76 • CID 2007:45 (1 July) • BRIEF REPORT
B R I E F R E P O R T
Fungal Burden, Early Fungicidal
Activity, and Outcome in Cryptococcal
Meningitis in Antiretroviral-Naive or
Treated with Amphotericin B or
Tihana Bicanic,1,4Graeme Meintjes,2,3Robin Wood,1Madeleine Hayes,4
Kevin Rebe,2,3Linda-Gail Bekker,1and Thomas Harrison1,4
1Desmond Tutu HIV Centre, Institute of Infectious Disease and Molecular
Service, GF Jooste Hospital, Cape Town, South Africa; and
Infectious Diseases, Department of Cellular and Molecular Medicine, St.
George’s University of London, London, United Kingdom
2Department of Medicine, University of Cape Town, and
(See the editorial commentary by Lortholary on pages 81–3)
In a prospective observational study of 54 patients with hu-
man immunodeficiency virus–associated cryptococcal men-
ingitis, the early fungicidal activity of amphotericin B (1 mg/
kg/day) was significantly greaterthanthatoffluconazole(400
mg/day). Compared with antiretroviral therapy–naive pa-
tients, patients developing cryptococcal meningitis while al-
ready receiving antiretroviraltherapyhadlowerbaselinefun-
gal burdens and a longer median duration of survival, but
there were no differences observed in fungal clearance, cer-
ebrospinal fluid proinflammatory cytokines, or 10-week
In African countries, prior to the availability of antiretroviral
therapy (ART), survival after experiencing cryptococcal men-
ingitis (CM) was poor, with an 88%–100% mortality rate at 6
months [1, 2]. Fluconazole was often the antifungal drug of
choice. The advent of ART altered the long-term prognosis for
patients with CM, although published datafromthedeveloping
Received 8 December 2006; accepted 21 February 2007; electronically published 25 May
Presented in part: 46th Interscience Conference of Antimicrobial Agents and Chemotherapy,
San Francisco, October 2006 (abstract 1768).
Reprints or correspondence: Dr. Tihana Bicanic, Div. of Infectious Diseases, Dept. of Cellular
and Molecular Medicine, St. George’s University of London, Cranmer Terrace, London SW17
ORE, United Kingdom (email@example.com).
Clinical Infectious Diseases2007;45:76–80
? 2007 by the Infectious Diseases Society of America. All rights reserved.
world are lacking. This neccesitates a fresh look at the benefits
and risks of implementing amphotericin B (AmB)–based ther-
apies in this context.
At Jooste Hospital (Cape Town, South Africa) in 2004, fol-
lowing expansion of the ART program,routineinitialtreatment
for CM was switched from 400 mg/day of fluconazole to 1 mg/
kg/day of AmB for 7 days, except for patients with a Glasgow
Coma Score of !10, who continued to receive fluconazole, and
except for times when the AmB supply was interruptedbecause
of a worldwide shortage of AmB . Access to ART may also
have altered the pathophysiology and clinical course of CM in
the proportion of patients now presenting with CM after ini-
tiating ART. We hypothesized that this group may mount a
work, we have demonstrated a correlation between such a pro-
inflammatory response and baseline fungal burden, clearance,
and outcome [4, 5].
Our aims were to determineandcomparetheearlyfungicidal
activity (EFA) and toxicity of AmB 1 mg/kg/day versus flu-
conazole 400 mg/day for the initial treatmentofHIV-associated
CM. In addition, we compared the results of analysisofbaseline
organism load, EFA, CSF immune response, and long-term
outcome among ART-naive and ART-experienced patients de-
This study was a prospective observational study
conducted at GF Jooste Hospital, and was approved by the
Research Ethics Committee of the University of Cape Town
(Cape Town, South Africa). From February to September2005,
after obtaining written informed consent from each person, we
enrolled 54 consecutive HIV-infected adults aged ?21 years
who were presenting with an episode of CM that had been
diagnosed using CSF India ink or positive CSF cryptococcal
antigen tests and confirmed with positive culture results for
Cryptococcus neoformans. First and relapse episodes of CM in
ART-naive and ART-experienced patients were included.There
were no exclusion criteria.
Patients were treated according to hospital protocol.Patients
with a baseline Glasgow Coma Score of ?10 received AmB
deoxycholate (Fungizone; Bristol-Myers Squibb) at a dosage of
1 mg/kg/day for 7 days, thereafter switching to oral fluconazole
(Diflucan; Pfizer) at a dosage of 400 mg/day for 8 weeks. Pa-
tients with a Glasgow Coma Score of !10 received fluconazole
at a dosage of 400 mg/day, from the outset, for 10 weeks. All
patients were switched to fluconazole at a dosage of 200 mg/
day after 10 weeks. In patients who were receivingconcomitant
rifampicin, the fluconazole dose was increased by 50% .
by guest on November 1, 2015
BRIEF REPORT • CID 2007:45 (1 July) • 77
Patients received 1 L of 0.9% saline solution daily, as well as
electrolyte supplementation as required. Follow-up lumbar
punctures were performed on days 3, 7, and 14. Patients with
an increased CSF opening pressure (125 cm of water) under-
went additional lumbar punctures, in accordance with current
guidelines . After patient discharge from the hospital, all
participants not already receiving ART received counselingand
initiated ART (stavudine plus lamivudine, plus efavirenz or
nevirapine) not sooner than 4 weeks after the startofantifungal
therapy. Patients were followed up for 1 year after enrollment
into the study.
Altered mental status was defined as any reduction in Glas-
gow Coma Score. All participants underwent baseline hema-
tological and biochemical testing, CD4 count and HIV viral
load were determined, and subsequent twice-weekly biochem-
for adverse effects of therapy. Quantitative fungal cultures of
CSF samples were performed, as described elsewhere . CSF
immune parameters (IFN-g, TNF-a, IL-1b, IL-2, IL-6 , IL-8,
IL-10, G-CSF, MCP-1, MIP-1a, MIP-1b, and Regulated upon
Activation, Normal T cell Expressed, and Secreted [RANTES]
levels) were determined using the Luminex multianalytesystem
(Luminex) and cytokine kits (Bio-Rad), as described elsewhere
, using a separate ELISA for IFN-g and TNF-a (Quantikine;
We compared baseline characteristics of groups using the x2
or Fisher’s exact test for categorical variables, and the Mann-
Whitney U test for continuous variables. Mortality at 10 weeks
and 1 year was compared using Fisher’s exact test. Survival
curves were compared using the Mantel-Haenszellog-ranktest.
EFA was compared between groups using linear regression [4,
5]. Median cytokine concentrations were compared using the
Mann-Whitney U test.
Sixty-one patients met the inclusion criteria, of
whom 54 gave informed consent and were enrolled. Table 1
shows baseline clinical and laboratory characteristics and out-
comes. Forty-nine patients (91%) were treated with AmB, and
5 received fluconazole. Fluconazole was administered to 2 pa-
tients because of a nonavailability of AmB, to 1 patient because
the patient refused hospital admission, to 1 patient because of
renal impairment (baseline creatinine level, 273 mmol/L), and
to 1 patient with a Glasgow Coma Score !10. The median
follow-up of survivors was 14 months.
In the AmB group, the median duration of treatment was 7
days (interquartile range, 6–8 days). Renal impairment (cre-
atinine level, 1220 mmol/L), developed in 4 patients. Only 1
patient required discontinuation of AmB before 7 days because
of renal impairment. No patients developed adverse events at-
tributed to fluconazole. The fluconazole-treated group showed
a trend towards more-severe disease: a higher baseline fungal
burden (340,000 vs. 74,000 colony-forming units (CFU)/mL
altered mental status (3 of 5 patients vs. 10 of 49;
and a higher viral load, comparedwiththeAmBgroup(420,000
vs. 44,000 copies/mL;).
P p .09
The EFA in patients treated with AmB was ?0.48?0.28
(mean ? SD) log CFU/mL of CSF per day (
calculated over the whole of the initial 2 weeks of therapy; this
value waswhen calculated over the first 7 days,
when patients were receiving AmB for all or most of the time.
Over 2 weeks, EFA was significantly higher for patients who
received AmB than for patients who received fluconazole as
initial therapy (EFA of fluconazole,
difference, 0.46 log CFU/day; 95% CI, 0.21–0.72;
(figure 1A and figure 1B). The difference remained significant
() if linear regression modeling was used to adjust for
P ? .005
baseline organism load and/or for baseline CSF IFN-g con-
centration—factors previously found to be associated with EFA
[4, 5]—or for plasma viral load, altered mental status, prior
receipt of ART, or if the experience was a first or a relapse
episode of CM.
Overall mortality was 17% at 2 weeks and 37% at 10 weeks.
Median survival was 110 days (153 days for AmB-treated pa-
tients and 61 days for patients treated initiallywithfluconazole;
) (figure 1C).
P p .03
In ART-naive patients, the median interval (interquartile
range) from initiating antifungal therapy to initiating ART was
46 (36–57) days. In ART-experienced patients, the median in-
terval (interquartile range) between initiating ART and pre-
senting to a health care facility with CM was 30 (21–77) days.
Compared with ART-naive patients, ART-experienced patients
had a lower baseline fungal burden (34,000 vs. 235,000 CFU/
mL CSF;), and displayed a trend towards higher WBC
P p .03
counts in CSF (24 vs. 7 cells/mm3;
There was no difference observed in EFA between the ART-
naive and ART-experienced groups treated with AmB (mean
even when linear regression modeling was used to adjust for
baseline organism load or baseline CSF IFN-g concentration.
When comparing outcome in ART-naive patients with ART-
experienced patients, 10-week mortality was similar (33% vs.
38%;), but 1-year mortality was significantly differentP p .8
(76% vs. 41%;) (table 1). Despite access to ART forP p .03
ART-naive patients after undergoing treatment of an initial
episode of CM, ART-experienced patients had a longer median
survival (11 year vs. 95 days;P p .04
no significant differences observed between ART-naive and
ART-experienced patients in the baseline CSF concentrations
of any of the 12 cytokines and chemokines measured (data not
shown), except for G-CSF (median level, 142 vs 307 pg/mL;
).P p .003
), a higher proportion of patientswhoexperienced
P p .06
P p .08
n p 49
P p .001
P p .19
P p .63
) (figure 1D). There were
by guest on November 1, 2015
Baseline clinical and laboratory characteristics and clinical outcomes, by drug treatment group and antiretroviral therapy (ART) status.
(n p 54)
Drug treatment group
(n p 49)
(n p 5)
(n p 36)
(n p 18)
No. (%) of male patients
Patient age, years
Mean weight ? SD, in kg
55 ? 12
56 ? 12
49 ? 3
54 ? 12
58 ? 12
No. (%) of patients with HIV infection status known at presentation
No. (%) of patients with abnormal mental status
CD4 count, cells ? 106/L
HIV load, copies/mL
No. (%) of patients who experience a relapse of CMa
No. (%) of patients receiving HAART
OP , cm H20
WBC count, cells/mm3
QC, CFU/mL CSF
Proportion (%) after 10 weeks of follow-up
Proportion (%) after 1 year of follow-up
Data are median (interquartile range), unless otherwise indicated. AmB, amphotericin B 1 mg/kg/day; CFU, colony-forming units; CM, cryptococcal meningitis; Flu, fluconazole; OP , opening
pressure; QC, quantitative culture.
aRelapse was defined as recurrent CM symptoms and positive CSF culture results in a patient with a documented, culture-confirmed prior episode of CM with complete clinical response to antifungal
treatment. Five of these patients were included in a prior report of the sensitivity of Cryptococcus neoformans isolates in cases of relapse . Patients were only included once, as either experiencing
a first episode or a relapse case.
bTwo patients were lost to follow-up at 10 weeks, and 3 patients were lost to follow-up at 1 year.
by guest on November 1, 2015
BRIEF REPORT • CID 2007:45 (1 July) • 79
CFU per mL of CSF per day was calculated for each patient using the slope of the linear regression of log CFU against time. For each treatment
group, early fungicidal activity (EFA) is shown as the mean ? SD rate of decrease in log CFU counts. EFA was significantly greater for amphotericin
B, compared with fluconazole ( ). C, Kaplan-Meier survival curves, from diagnosis of cryptococcal meningitis (CM), by drug treatment group.P p .001
Median survival was significantly greater for amphotericin B than for fluconazole (153 days vs. 61 days;
survival curves from cryptococcal meningitis diagnosis by antiretroviral therapy (ART) status at presentation. Median survival was significantly greater
for patients who presented while receiving ART (11 year vs. 95 days for those not receiving ART;
A and B, the decrease in CSF Cryptococcus neoformans colony-forming units (CFU) over time, by treatment group. The decrease in log
with the x2test). D, Kaplan-MeierP p .03
with the x2test). CM, cryptococcalP p .04
patients with more-severe disease receiving fluconazole, means
that differences in median survival between treatment groups
may be subject to bias. However, the marked difference in EFA
of fluconazole and AmB, which was significant even when
markers of severity of disease or host immunity shown to be
associated with clearance were controlled for [4, 5], likely rep-
resents a real difference in the activity of the drugs. At a dosage
of 400 mg/day, fluconazole dosage is almost static over the first
2 weeks of therapy.
Given a previous study linking CSF proinflammatory cyto-
kines—IFN-g in particular—to EFA , the lack of any dif-
ference in CSF immune parameters between ART-naive and
ART-experienced patients (except for G-CSF, a finding that
needs to be confirmed in an independent series) is consistent
with the fact that we also found no difference in EFA between
these 2 groups. There is ongoing debate as to the risks and
benefits of immediate or early institution of ART in the setting
of acute CM in ART-naive patients. Our findings of similar
acute CM-related mortality but large differences in 1-year sur-
vival between ART-naive and ART–experienced patients pre-
senting with CM would suggest that such a strategy may not
reduce acute CM-related deaths, but may reduce deaths due to
other HIV infection–related causes. The risks of administration
of earlier ART, particularly of increased rates and severity of
immune reconstitution reactions [8–10], remains unclear.
It is interesting to note that a recent study  found no
evidence for a decrease in acute (3-month) mortality among
patients presenting with CM in France in the ART era, com-
pared with the pre-ART era. However, in that study, after the
introduction of ART, only 21% of French patients with CM
were receiving ART at the time of CM diagnosis, compared
with 100% of the ART-experienced group who presented with
CM in our study.
The marked difference in EFA and survivalinpatientstreated
by guest on November 1, 2015
80 • CID 2007:45 (1 July) • BRIEF REPORT Download full-text
with AmB versus fluconazole lends further support to the use
of AmB to treat CM wherever feasible, even if resources limit
the duration of AmB to !2 weeks. At 1mg/kg/day, AmB was
very well tolerated in this setting. When safe adminstration of
AmB is not possible, these results underline the urgent need
for studies, currently underway, to determine the efficacy of
higher doses of fluconazole. This study confirms that, when
acute infection has ended and an ART regimen has been es-
tablished, patients in resource-limited areas who have HIV in-
fection–associated CM have a good prognosis. The challenge
remains to improve acute disease management, thereby in-
creasing the proportion of patients who survive the critical
Sister Nomqondiso Sidibana was the research nurse and the Xhosa in-
terpreter for the study. We wish to particularly thank all of the patients
participating in this study. We also thank the doctors, nurses, and HIV
counselors at the GF Jooste Hospital (Cape Town, South Africa); the med-
ical wards, the Carnation ward, and the infectious diseases clinic; Anthony
Williams and other staff at the GF Jooste laboratory (Cape Town); and the
staff at the Desmond Tutu HIV Centre at the University of Cape Town
(Cape Town) for their support and assistance in conducting this study.
British Infection Society (fellowship grant to T.B.).
Potential conflicts of interest.
All authors: no conflicts.
1. Mwaba P. Mwansa J, Chintu C, et al. Clinical presentation, natural
history, and cumulative death rates of 230 adults with primary cryp-
tococcal meningitis in Zambian AIDS patients treated under localcon-
ditions. Postgrad Med J 2001;77:769–73.
2. Mayanja-Kizza H, Oishi K, Mitarai S, et al. Combination therapy with
fluconazole and flucytosine for cryptococcal meningitis in Ugandan
patients with AIDS. Clin Infect Dis 1998;26:1362–6.
3. Bicanic T, Wood R, Bekker LG, et al. Antiretroviral rollout, antifungal
roll-back: access to treatment for cryptococcalmeningitis.LancetInfect
4. Brouwer AE, Rajanuwong A, Chierakul W, et al. Combination anti-
fungal therapies for HIV-associated cryptococcal meningitis: a ran-
domised trial. Lancet 2004;363:1764–7.
5. Siddiqui A, Brouwer AE, Wuthiekanun V, et al. IFN-gamma at the site
of infection determines rate of clearance of infection in cryptococcal
meningitis. J Immunol 2005;174:1746–50.
6. Bicanic T, Harrison T, Niepieklo A, et al. Symptomatic relapse of HIV-
associated cryptococcal meningitis after initial fluconazole monother-
apy: the role of fluconazole resistance and immune reconstitution.Clin
Infect Dis 2006;43:1069–73.
7. Saag MS, Graybill RJ, Larsen RA, et al. for the MSG cryptococcal
subproject. Practice guidelines for the management of cryptococcal
disease. Clin Infect Dis 2000;30:710–8.
8. Shelburne SA 3rd, Darcourt J, White AC Jr, et al. The role of immune
reconstitution inflammatory syndrome in AIDS-related Cryptococcus
neoformans disease in the era of highly active antiretroviral therapy.
Clin Infect Dis 2005;40:1049–52.
9. Lortholary O, Fontanet A, Memain N, et al. Incidence and risk factors
of immune reconstitution inflammatory syndrome complicatingHIV-
associated cryptococcosis in France. French Cryptococcosis Study
Group. AIDS 2005;19:1043–9.
10. Lawn SD, Bekker LG, Myer L, Orrell C, Wood R. Cryptococcocal
immune reconstitution disease: a major cause of early mortality in a
South African antiretroviral programme. AIDS 2005;19:2050–2.
11. Lortholary O, Poizat G, Zeller V et al. Long-term outcome of AIDS-
associated cryptococcosis in the era of combination antiretroviralther-
apy. AIDS 2006;20:2183–91.
by guest on November 1, 2015