Cryptococcal meningitis poses an ongoing public health burden in Africa, particularly among those with HIV/AIDS. New diagnostic tools like the lateral flow immunoassay for cryptococcal antigen detection in urine could enable earlier diagnosis and treatment. Ongoing clinical trials are evaluating shorter courses of amphotericin B combined with high-dose fluconazole as alternative treatment strategies that are more feasible and sustainable in resource-limited settings.

1. Confronting Cryptococcal meningitis in Africa:
New diagnostic, prevention and treatment strategies
to reduce disease burden
Tom Harrison
Meningitis Research Foundation
November 5th, 2013

2. Plan:
• Epidemiology
• ongoing burden HIV-associated CM (non HIV CM)
• Diagnostics
• New LF point-of-care dipstick test for CrAg
• Use in screening, early diagnosis
• Therapy
• Current options:
• Developed and Resource-limited settings
• Ongoing and future studies
• [Mnx of complications: CM-IRIS, timing ART (COAT study),
raised CSF pressure. Immunology of HIV-CM, virulence &
evolution C. neoformans]

3. HIV-associated cryptococcal infection:
• Accounts majority global burden of CM
• Cryptococcal meningitis: common in late stage HIV
• Treatment is poor – > 50% 10 wk mortality in Africa
• Common cause of death in HIV cohorts - around 15% all
HIV deaths S/E Africa
• At least 100,000 deaths/yr in SSA: CDC – CID 2009; 23:525
• Not decreasing yet with ART in most centres
• But ART means good long term prognosis if survive
acute infection

4. Aetiology of Adult Meningitis in S/E Africa
Cape Town, South Africa
Jarvis J et al. BMC Inf Dis 2010
Kampala, Uganda
Durski K et al. JAIDS 2013

5. S Africa: high CM burden despite ART rollout
Source: National Institute for Communicable Diseases, Johannesburg, SA

6. Bicanic et al CROI 2011 P-150, Submitted

7. • Problem: Late presentation. Need: Earlier Diagnosis
• Early features – headache, fever v. non specific – patients
often not referred immediately to hospital for LP – by
which time may be too late for effective Rx
• Need Point-of-Care diagnostic test
• Polysaccharide capsule - major virulence factor but also
?Achilles heel of Cryptococcus
basis specific, early (subclinical stage) diagnostic test
(Latex agglutination assay CrAg)
• POC format
Immuno-Mycologics (Sean Baumann, CEO),
Tom Kozel (Univ. of Nevada at Reno):

8. LFI dipstick for GXM.
LFI was constructed
using a cocktail of
mAbs F12D2 and 339
KOZEL LAB.
The assay has a
sensitivity of 2-5
ng/ml for serotype A
GXM:
Jarvis et al CID
2011; 53:1019
Table 3. Sensitivity limits of a prototype LFI assay (constructed by IMMY) for
GXM of serotypes A, B, C and D compared with commercially available latex
agglutination assaysa
Immunoassay
Serotype Ab
Serotype B
Serotype C
Serotype D
CN6
MU-1
184
409
2406
6
KC
298
9375
127
Prototype LFI
assay
2.4
5.0
4.6
3.0
16
7.0
10
5.1
Commercial
latex
agglutinationc
250
500
500
250
8,000
4,000
1000
250

9. Crag Lateral Flow Assay

10. CrAg LFA test for serum, CSF (samples already approved
for latex agglutination test) approved in Europe, and now
by FDA
But, ideally need test using non-invasive sample, with no
processing
Urine
Drop whole blood

11. 106
Serum vs. plasma
104
Plasma vs. urine
104
105
103
103
104
102
Serum vs. urine
102
103
104
105
Serum (ng/ml)
106
R = 0.97
P < 0.0001
Slope = 0.097
101
100
100
10
1
10
2
10
3
10
4
Plasma (ng/ml)
10
5
Urine (ng/ml)
102
102
Urine (ng/ml)
R = 0.997
P < 0.0001
Slope = 0.85
103
10
6
R = 0.98
P < 0.0001
Slope = 0.083
101
100
100
101
102
103
Serum (ng/ml)
Concentration of GXM in paired serum, plasma and urine
from 25 subjects.
Blood and urine were collected from 25 patients in Cape
Town with culture proven cryptococcosis. Serum, plasma
and urine were evaluated by quantitative ELISA to assess
concentrations of GXM.
Jarvis et al CID 2011; 53:1019
104
105
106

12. Sensitivity of Urine vs Plasma/Serum LFA
in culture-confirmed cases CM - Cape Town
Retrospective Study:
Cryptococcus culture-confirmed paired serum, plasma and urine specimens
from 62 South African patients in Cape Town
Serum
Plasma
Urine
Crag LFA (+)
61
61
61
Crag LFA (+/-)
1
1
0
Crag LFA (-)
0
0
1
Crag LFA
Sensitivity
95% CI
Serum
Plasma
Urine
100%
100%
98%
94-100%
94-100%
91-100%
Jarvis et al CID 2011; 53:1019

13. • POC could be used:
• Facilitate earlier diagnosis (in primary care settings) and
treatment all symptomatic cases
• Prevention clinical CM by screening for subclinical
infection and pre-emptive treatment
• Targeted use pre-emptive fluconazole therapy for patients
with CD4<100 who are CrAg positive, after HIV diagnosis
and prior to starting ART:
an alternative strategy to primary “across-the-board”
fluconazole prophylaxis
Jarvis et al: Clin Infect Dis 2009; 48:856

14. • In South Africa
• 20-33% of cases now present after initiation of ART
• Over 70% present after a diagnosis of HIV
Bicanic CID 2007; 45:76 Jarvis CID 2010; 51:1463
• All these cases are potentially preventable
• Screening identifies those patients at risk :
Jarvis et al: Clin Infect Dis 2009; 48:856
Serum Antigen positive (approx 5-10% of CD4<100)
28% developed meningitis
Serum Antigen negative (90%+ of CD4<100)
0/660 patients developed meningitis

15. Current Gold Standard Therapy:
Amphotericin B plus Flucytosine
IDSA Perfect et al CID 2010 50:291-322,
WHO Rapid advice guideline, WHO November, 2011:
2 wks:
AmB 0.7-1 mg/kg/d plus flucytosine (5-FC) 100 mg/kg/d
Based on..Van der Horst et al NEJM 1997; 337:15
Supported by…Brouwer et al: Lancet 2004; 363:1764-67
Nailed by….Day et al NEJM 2013; 368:1291-302

16. • Vietnam trial, Day et al
Induction with:
•
AmB 1 mg/kg/d alone, 4 weeks
• Vs AmB 1 mg/kg/d alone plus 5FC 100 mg/kg/d, 2 weeks
• Vs AmB 1 mg/kg/d alone plus fluconazole 800 mg/d, 2
weeks
• To clinical endpoints [n=300]
• Results presented ICAAC last year, NEJM April 2013

17. Vietnam trial, Jeremy Day et al [ISRCTN95123928]
AmB+5FC
AmB
+flucon
AmB alone
Day J et al ICAAC 2011

18. Amphotericin B combinations
Given availability (low or no cost), safety fluconazole, but
current lack access 5FC
Important question for high burden countries in Africa in
particular is:
Is AmB plus fluconazole preferable to AmB alone?

19. Vietnam trial, Jeremy Day et al
Day J et al N Engl J Med 2013;368:1291-302

20. Southern African J HIV Med 2013; 14:76
Vietnam trial plus
Pappas et al: CID 2009; 48 1775-83
in the absence of 5FC…
AmB 1 mg/kg/d plus fluconazole 800 mg/d

21. Antifungal therapy: New Paradigms in resource-restricted
settings
Problem
1. accepted gold standard 2 wks AmB+5FC not available,
feasible in many African centres
2. currently widely-used and available alternative oral
fluconazole - inadequate

22. F
S
C
Fluconazole 400 mg/d
l
m
/
U
F
C
Log CFU/ml CSF
7
6
5
4
3
g
2
o
L 1
0
0
2
4
6
8
Day
10 12 14
Amphotericin B 1 mg/kg/d
7
6
5
4
3
2
1
0
0
2
4
6
8
Day
Bicanic T et al, Clin Infect Dis 2007; 45:76-80.
10 12 14

23. Fluconazole 800
EFA -0.08 logCFU/d
Log CFU/ml CSF
7
No increased toxicity observed
– small numbers
6
5
4
Longley, Muzoora, et al
Clin Infect Dis 2008; 47 1556-61
3
2
1
2
4
6
10 12 14 16
Day
Fluconazole 1200
EFA -0.18 logCFU/d
7
Log CFU/ml CSF
8
6
5
0.5
Patients
from Cape
Town
0.0
4
3
2
-0.5
1
0
Rate of clearance (log CFU/d)
0
2
4
6
8
10 12 14 16
Day
400
800
1200
Fluconazole dose (mg/d)

24. Nussbaum et al. Clin Infect Dis 2010; 50:338-44
UNC Project, Lilongwe, Malawi

25. 1 .0 0
0 .8 0
s u r v iv a l p r o b a b ilit y
0 .6 0
0 .4 0
0 .2 0
0 .0 0
F lu c o n a z o le m o n o t h e r a p y
F lu c o n a z o le / 5 F C c o m b in a t io n
0
2
4
6
8
n u m b e r o f w e e k s f o l lo w - u p
2 week mortality: HR 0.24 (0.05-1.16) p=0.05
Nussbaum et al. Clin Infect Dis 2010; 50:338-44
10

26. Alternative, resource-restricted centres:
Add Short course AmB (5-7 days)
Cohorts in Mbarara, Uganda (5 d) and Lilongwe, Malawi (7d)
(Muzoora et al J.Infect 2012, Jackson et al, AIDS 2012)
•Very large gain:
Tolerability
Feasibility, sustainability
•?Would efficacy be compromised?
Large initial reduction in organism load
Carries on through second week - with no flattening
?long half life AmB
Follow up therapy: high dose fluconazole

27. Muzoora et al: J Infect 2012; 64:76
30 patients Rx 1200 mg/d flucanozole plus AmB 1 mg/kg/d 5 days:
EFA -0.30 log CFU/d over 2 weeks, -0.31 over 7 days

28. Gain in tolerability::
Muzoora et al
Flu1200+ AmB1.0 5d
Laboratory parameter
Decrease in hemoglobin
level,
>2 g/d, (%)
Decrease in hemoglobin
level,
% change, mean
Creatinine level >2x
baseline level (%)
AmB 2 wks Bicanic
CID 2008; 47:123 -30
Day 14
Day 14
AmB 0.7
AmB 1.0
Day 7
Day 14
17
10
50
71
8
6
16
25
4
4.5
13
32
no grade III/IV anemia, ↑ALT,
no grade IV hypokalaemia, ↑creatinine
Trends in mortality: ↓ compared earlier Mbarara
cohorts: 2 and 10 wks: 23%, 28%

29. ACTA Trial: MRC funded: MLW Blantyre, UNC
Lilongwe, UTH Lusaka; ANRS: Cameroon
Strategy 1: Fluconazole 1200 mg /d plus flucytosine 25
mg/kg qds for 2weeks.
Strategy 2: Amphotericin B (AmB) 1 mg/kg/d +
fluconazole 1200 mg /d OR 5FC for 7 days
Strategy 3: Amphotericin B (AmB) 1 mg/kg/d +
fluconazole 1200 mg /d OR 5FC for 14 days

30. Potential Impact
Strategies 1, 2, much more readily, safely sustained than 3.
IF either shown to be as effective as 3, could result in a
reduction in the 10-week mortality in resource-limited
settings using fluconazole from around 50-60%
to the 30-35% seen with 2 wk AmB-combination treatment.
IF NOT as effective then substantial extra resources to
SAFELY deliver 2 wks AmB-combination Rx are justified

31. Other ongoing studies
Adjunctive Steroid study CRYPTODEX (Jeremy Day,
David Lalloo and colleagues)
ACTG 5225: Fluconazole dose escalation (1200g2000g/d) vs AmB. (Bob Larsen and colleagues)

32. Other planned / ongoing studies
Phase II: 3rd Strategy building on high dose fluconazole
backbone – adding
Intermittent high dose Ambisome – 1, 2, or 3 doses, vs
standard daily dosing Ambisome, with EFA endpoint.
Joe Jarvis, et al. Mwanza Tanzania.

33. CMAG - cryptococcal meningitis action group
Loyse A et al. Lancet Infect Dis. 2013 Jul;13(7):629-37
GAFFI – Global Action For Fungal Infections - David Denning
and colleagues
AmB and 5FC from 2013 on WHO
essential medicines list

34. St. George’s
Tihana Bicanic
Joe Jarvis
Angela Loyse
Nicky Longley
Sile Molloy
Annemarie Brouwer
Thailand
Nick White, Nick Day
Funders:
Wellcome Trust
MRC
USAID
DFID Malawi
British Infection Society
Lancet
ANRS
South Africa
Graeme Meintjes,
Linda-Gail Bekker, Robin Wood
Mbarara, Uganda
Conrad Muzoora, Kabanda Taseera
Lilongwe Malawi, UNC Project
Jesse Nussbaum, Charles Van der Horst, Dan
Namarika, Mina Hosseinipour
Blantyre, MLW, CoM: Rob Heyderman, David
Lalloo, Kate Gaskell
Lusaka UTH: Shabir Lakhi, Duncan Chanda
Cameroon: Charles Kuouanfack, Elvis Temfack
Pasteur Institute: Olivier Lortholary
London School Hygiene Tropical Medicine
Shabbar Jaffar, John Bradley
University Nevada, Reno: Tom Kozel
Immuno-Mycologics: Sean Baumann