P. falciparum resistance in Nigeria

Christian T. Happi

Malaria in Nigeria: Facts and Figures

• Population:160 millions

• 350,000 Nigerian children under the age of 5 die

of malaria every year [2002 Annual report of the

Federal Ministry of Health (FMOH), Nigeria]

• Drug resistance is a growing problem

Current situation of drug resistant malaria in Nigeria

CLINICAL PROFILES OF CURRENTLY USED ANTIMALARIAL DRUGS IN NIGERIA

• CHLOROQUINE:

– DATE CURE RATE

– 1990 100%

– 1992 85%

– 2003 48%

– 2004 43%

• SULPHADOXINE/PYRIMETHAMINE

– DATE CURE RATE

– 1990 100%

– 1993 93%

– 1997 80%

– 2003 77%

– 2005 73%

• HALOFANTHRINE

– DATE CURE RATES

– 1990 100%

– 1997 87%

• Innate resistance to mefloquine

STUDY SITE/DESIGN AND METHODOLOGY

Study site:

-Malaria Clinic, University

College Hospital

Clinical studies:•Study received ethical approval from the joint UI/UCH and the Harvard Schoo

of Public Health ethical review committee.

Study Design

Patients enrollment

AS-AQ Artemether-Lumefantrine

Samples collectionSamples collection

Nested PCRIn vitro

susceptibility testing

Nested PCR

Msp-2 typing

Pfcrt, Pfmdr1,

Transporters

Msp2 typing

RFLP

Pfmdr1, Pfatpase6, transporter

genes

RFLP

Patient treatment

outcomes

Patient treatment

outcomes

Cloning and

sequencing of

PCR Products

PKPK

In vitro susceptibility

testing

Determination of P. falciparum population structure

and discrimination between recrudescences and re-

infections

• Nested PCR using block 3 of msp-2 gene.

• Resolve secondary products on 1.5% agarose gels stained with ethidium bromide.

• View and photographed on a UV transilluminator.

L D0 D21 D0 D3 D14 D0 D14 D0 D28 D0 D7

COMPARATIVE IN VITRO SENSITIVITY PROFILES OF PATIENT

ISOLATES OF P. FALCIPARUM TO ANTIMALARIAL DRUGS

0

20

40

60

80

100

Percen

t CQ SENSITIVE

AQ SENSITIVE

QN SENSITIVE

SP SENSITIVE

Association between pfcrt and pfmdr1 alleles and in vivo

CQ resistance

477Number

failed

80%62%49%87%0.00042

2928Number

cured

pfmdr1Y86pfmdr1N86

49 6 Number

failed

73%55%28%89%0.0196

40 16 Number

cured

NPVPPVSpecificitySensitivityP value

pfcrtT76pfcrtK76Treatment

outcome

Genotypes of

pre-treatment

isolates

pfcrt T76 (%) Pfcrt K76 (%) Total

Pfmdr1Y86 26 (47%) 5 (9%) 31

Pfmdr1N86 14 (26%) 10 (18%) 24

Total 40 15 55

Distribution of mutant pfcrt and pfmdr1 alleles among the 55 pre-

treatment isolates harboring a single allele on both loci

χ2= 4.45; p=0.0349.

D’ (95% CI) r2 p

Paired alleles pfcrt T76 pfcrtK76

pfmdr1Y86 26 14

0.64

(0.537-0.753)0.28 0.000

Pfmdr1N86 5 10

Association and linkage disequilibrium between paired single alleles

of pfcrt on Chromosome 7 and pfmdr1 on Chromosome 5.

Selection of mutant pfmdr1Y86 alleles in Nigerian

children by CQ treatment

5322724Total

10343Mixed

Allele

7511Wild-type

N86

3614220Mutant Y86

Recrudescence

Mixed

Allele

Wild-type

N86

Mutant

Y86

Pfmdr1

Codon 86

TotalEnrollment

p=0.006 (Wilcoxon Signed Ranks Test); Z=-2.727 (based on positive Ranks)

Selection of mutant pfcrtT76 alleles in Nigerian

children by CQ treatment

5314633Total

8413Mixed

Allele

2020Wild-type

K76

4310330Mutant T76

Recrudescence

Mixed

Allele

Wild-type

K76

Mutant

T76

pfcrt Codon

76

TotalEnrollment

p=0.040 (Wilcoxon Signed Ranks Test); Z=-2.051 (based on positive Ranks)

COMPARISON OF CLINICAL OUTCOME WITH BLOOD LEVELS OF

CHLOROQUINE AND AREA UNDER CONCENTRATION TIME CURVE (AUC)

AND CLEARANCE

25.2±22.2449.0±379.42.7±1.70.932±0.481.56±0.880.80±0.52LTF(30.2%)

P=0.20

1.8±0.7

3.8±3.0

Mean

Cmax

(ug/ml)

P=0.79P=0.36P=0.28P=0.09P = 0.72

26.21±26.9320.9±195.60.92±0.991.20±0.770.58±0.60ETF(26.8%)

20.03±11.1445.2±213.21.57±1.482.10±1.190.70±0.65ACR (43%)

D7D3D0

Mean

Clearance

(mlday-1kg-1)

x1000

Mean AUC

(ugdayml-1)

Mean Concentration of CQ (ug/ml) Clinical

outcome

(%patients)

Concentration-time curve for patients treated with CQ

0 4 8 120.0

0.5

1.0

1.5

2.0

2.5ACR

ETF

LTF

Day

Concentr

ation (ug/m

l)

0 3 7 14 21 280

250

500

750Blood CQ conc

CQR Gametocytes

0

30

60

90

CQS gametocytes

92% pfcrtT7678% pfmdr1Y86

MEC

Time (days)

[CQ

]b

loo

d

Ga

me

toc

yte

s d

en

sity

/ul b

loo

d

The median in vivo MEC of CQ was 780nM (250ng/ml)[range: 20.7-1400ng/ml] in

blood from patients. Blood CQ concentration on day 7 post-treatment (during peak

gametocytaemia) was above therapeutic concentration (115 -263 ng/ml) in all patients.

Gametocytes Generation, Blood levels of CQ and Molecular Markers

of Chloroquine Resistance

0 3 7 14 21 280

2

4

6 CQR MSP2

CQ blood conc

CQS MSP-2

0

100

200

300

400

500

600

M E C

92% pfcrt T76

78%pfmdr1Y86

Time (days)

Me

an

no

. c

lon

es

pe

r in

fec

tio

n

[CQ

]blo

od (n

g/m

l)

Gametocytes Generation, Blood levels of CQ and Molecular Markers

of Chloroquine Resistance

Association between molecular markers of

SP resistance and patients treatment

outcome.

0

20

40

60

80

100

Pe

rce

nt

Asn-108

Ile-5

1Arg

-59

Gly

-437

Glu

-540

Asn-108

/Ile5

1/Arg

59

Gly

-437

/Glu

540

quintu

ple d

hfr/dhps

muta

nt

Prevalence of dhfr and dhps mutations at enrollment in isolates of Pf

36.80 (9.28-146.19)

3.28 (1.77- 6.09)

0.000*

0.002

17 (74%)

7 (100%)

4 (7%)

7 (30%)

Quintuple dhfr/dhps

mutant

age ≤ 5 years

age ≥ 5 years

224 (24-2034.28)

1.64 (1.18-2.28)

0.000*

0.071

22 (96%)

7 (100%)

5 (9%)

14 (61%)

Triple dhfr mutant

age ≤ 5 years

age ≥ 5 years

OR (95% CI)P valueFailed

(%)

Cured

(%)

dhfr/dhps mutations

and age stratification

Treatment outcome

Association betweem dhfr/dhps mutations and treatment outcome

Genotype failure Index

Based on quintuple mutant

• GFI= 1.07 (29%/27%)

Molecular markers of amodiaquine resistance

Baseline amodiaquine in vivo and in vitro sensitivity

87

13

76.9

23.1

0

10

20

30

40

50

60

70

80

90

Pe

rce

nt

Cured Failed In vitro

sensitive

In vitro

Resistant

Baseline frequency of pfcrt and pfmdr1 in P.

falciparum from amodiaquine treated patients

49.5

18.28.1 15.2

34.3

22.2

0

25

50

75

T76 K76 K76+T76 Y86 N86 N86+Y86

Treatment outcome (%)

pfcrt/pfmdr1alleles Failed Cured OR (95% CI) p-value

pfcrt

T76a 13/13

(100%)

67/88 (76%)1.31 (1.16-1.47) 0.065

K76b 0/13 (0%) 21/88 (24%)

pfmdr1

Y86c 11/13 (85%) 46/88 (52%)5.022 (1.051-

23.98)0.036‡

N86d 2/13 (15%) 42/88 (48%)

pfmdr1 & pfcrt

pfmdr1Y86+ pfcrtT76 11/13 (85%) 37/88 (42%)7.58 (1.58-36.25) 0.006‡

pfmdr1N86+ pfcrtT76 2/13 (15%) 51/88 (58%)

Association between pfcrt/pfmdr1 mutations in P. falciparum

isolates collected at enrolment and AQ treatment failure.

Prevalence of point mutations in patients

samples (n)

Pfmdr1/pfcrt alleles Baseline Recrudescence χ2 (Chi-square) p-value

Pfmdr1 Codon 86

Wild-type N86 43% (44/101) 8% (1/13) 5.91 0.013*

Mutant Y86 29% (29/101) 84% (11/13) 13.44 0.000246*

Mixed N86+ Y86 28% (28/101) 8% (1/13) 1.49 0.221

pfcrt Codon 76

Wild-type K76 22% (22/101) 0% (0/13) 2.25 0.133

mutant T76 62% (63/101) 92% (12/13) 3.31 0.0671

Mixed K76+T76 16% (16/101) 8% (1/13) 0.13 0.716

Pfmdr1 Codon 86+ pfcrt Codon 76

N86+K76 12% (12/101) 0/% (0/13) 0.70 0.404

Y86+T76 46% (47/101) 92% (12/13) 10.08 0.0015*

Mixed (N86+T76/Y86+K76) 42% (42/101) 8% (1/13) 4.28 0.038*

Selection of mutant pfcrtT76 and pfmdr1Y86 alleles in Nigerian children by amodiaquine treatment

Patients clearing

double mutant

parasites

(n)

Patients not

clearing

double mutant

parasites

(n)

p

value

Mean age in years 6.42±2.57 (37) 3.90±2.57 (11)

0.0017*

Fever 89.2% (33) 45.4% (5)

0.0066*

Mean log of parasites

density

10.25±0.93 9.96±1.180.4

Association between patients’ characteristics and clearance of amodiaquine resistant P. falciparum§ in children from Nigeria

Confirmation of emergence of mutations associated with

Atovaquone/proguanil resistance in unexposed Plasmodium

falciparum isolates from Nigeria.

150bp

174bp

1 2 3 4 5 6 7 8 9 10

Cytb011 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKMPFYPNLL 205Cytb012 GYTVSDXTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb014 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb023 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb024 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb027 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb028 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb029 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb038 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb041 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb043 GYTVSDPTLKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb053 GYTVSDPTLKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb053d10 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb057 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb059 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205K1F GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205NGTV01 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 2053D7 GYTVSDPTIKRFFVLHFILPFIGLCIVFIHIFFLHLHGSTNPLGYDTALKIPFYPNLL 205Cytb011 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFNNNNAMLKTVPSKPA 280Cytb012 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLTFYAMLKTVPSKPA 280Cytb14F SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280Cytb023 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280Cytb024 SLDVKGFNNVIILFLTQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWHFLPFNNNNAMLKTVPSKPA 280Cytb027 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFNNNNAMLKTVPSKPA 280Cytb028 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280Cytb028 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280Cytb038 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFNNNNAMLKTVPSKPA 280Cytb041 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280Cytb043 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280Cytb053 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280Cytb053d10 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFNNNNAMLKTVPSKPA 280Cytb057 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280Cytb059 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280K1F SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280NGTV01 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFNNNNAMLKTVPSKPA 2803D7 SLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVVNTYVTPSQIVPEWYFLPFYAMLKTVPSKPA 280

Summary (1)•Resistance level of Plasmodium falciparum to Chloroquine or SP is on the increase.

•In vitro results shows concurrent reduced susceptibility to currently available antimalarial drugs including amodiaquine which is the drug to be combined with artesunate in the recently reviewed malaria control policy of Nigeria.

•A significant number of patients had chloroquine in blood samples prior to treatment however this did not appear to influence the response to treatment.

•Preliminary pharmacokinetic data generated appears to show no significant difference in the area under the concentration time curve (AUC) (which is a measure of exposure to drug) and blood levels on day 7 from patients who were cured or who failed chloroquine treatment.

•Mutations in pfcrt and pfmdr1 genes are linkage disequilibrium and this linkage is maintained epistatically through selection by chloroquine

Summary (2)

•Early detection of gametocytes in patients isolates may be a good indicator of parasites resistance to CQ.

•Gametocytes harboring the mutant pfcrt T76 and pfmdr1Y86 alleles may have a relative survival advantage in presence of high plasma concentrations of CQ.

•The dhfr/dhps quintuple mutants is the best predictive marker of S-P resistance

•pfcrtT76 and pfmdr1Y86 appear to be good molecular markers of amodiaquine resistance.

•The detection of atovaquone resistance associated mutations in unexposed populations of P. falciparum in Nigeria is a matter of serious concern

•Data from the project were used to promote evidence-based antimalarial drug policy in Nigeria.

The Way Forward

• Identification of new molecular markers of resistance to

artemisinin derivatives

• Identification of immune determinants of clearance of

Drug resistant parasites

Identification of new molecular markers of resistance to artemisinin

derivatives

Transporters

• G2-PF-A0590w (3),

• G7-PF13-0271 (1),

• G25-PF14-0321 (2)

• G70-PFL0620c ( 1)

• G30-PF14-0292 (1)

• G47-PFE0775c (3),

• G49-PF08-0078 (8),

• G54-PF14-0260 (2)

• G55-PF14-0133 (1)

pfcrt

Pfmdr1 (86, 1042, 1246

• PfATP6

Blood Sample

Total RNAPlasma DNACulture

•Drug Blood Levels

(specific aim 1)

In vitro Susceptibility Test

(specific aim 1)

•P. falciparum population diversity

[MSP2,microsatellites]

•SNPs Transporters genes

(specific aim 2)

•Transporters gene expression

Using both Quantitative RT PCR

and customized microarrays

(specific aim 3)

•Parasite Cloning

(specific aim 3)

•Transporter gene expression

Fresh field isolates vs Lab clones

(specific aim 4)

Preliminary data

• In vitro development of a stable artemisinin

Plasmodium falciparum Resistant Clone (W2-

ART).

- survives a 200ng/ml [artelinic acid] in vitro

-phenotype is stable

-being characterized for SNPs and for whole

genome scan (Real-time QT PCR) and

microarrays

Selection of N86 allele of pfmdr1 by Artemether-

lumefantrine

28.6

71.4

50

21.4 21.4

7.2

0

10

20

30

40

50

60

70

80

N DY Y

D0

Drec/reinf

P=0.01; z=-2.53 Wilcoxon-signed rank test

Selection of F184 allele of pfmdr1 by

Artemether-lumefantrine

78.6

14.3 14.3 14.3

7.1

71.4

0

10

20

30

40

50

60

70

80

Y YF F

D0

Drec/reinf

P=0.003; z=-2.97; Wilcoxon-signed rank test

Selection of D1246 allele of pfmdr1 by Artemether-lumefantrine

71.4

85.7

21.4

0

7.114.3

0

10

20

30

40

50

60

70

80

90

D DY Y

D0

Drec/reinf

P=0.7; Wilcoxon singed ranked