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Clinical and Laboratory Assessment of Antimalarial Drug Efficacy

in the Lao P.D.R

Presentation Plan 1.  Introduction

2.  Studies conducted before 2000 (in-vivo)

3.  Studies after 2000:

- In-vitro study

- Molecular study

- Clinical trials

Abbreviations

CQ = Chloroquine

SP = Fansidar

QN = Quinine

A = Artesunate

M = Mefloquine

AL = Artemether-lumefantrine (Coartem)

DP = Dihydroartemisinin-piperaquine

(Artekin)

Before 2005… •  Malaria: important cause of morbidity & mortality in Laos (Prevalence ~ 14%)

•  Chloroquine (CQ) & sulfadoxine-pyrimethamine (SP): did not work anymore in neighboring countries

•  But CQ & SP: Lao nationally recommended antimalarials for treatment of uncomplicated malaria !

A Lao child with severe malaria

A Lao technician said in 2000:

“…..in Laos, chloroquine still works OK and

there is no resistance of malaria parasites. If

you don’t believe me, let’s see in your

study….”

Efficacy of Antimalarial Drugs in Laos before 2000

1.  Ebisawa et al., 1970 (Japan J Exp Med 40: 151-157)

-  Antimalarial drugs: CQ, SP, QN + SP -  Nam-Ngum dam, Vientiane (1968); N = 108

Results CQ SP QN+SP (n=64) (n=26) (n=18)

Sensitive 59 (92%) 25 (96%) 18 (100%)

RI 5 (8%) 1 (4%) 0

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2.  AL Tawil, 1977 (Bull WHO: 230-237)

-  Antimalarial drug: CQ

-  Nam-Ngum dam, Vientiane (1975-76)

-  N = 48 (follow up: 7 days) Results Sensible = 28 (58%)

RI = 8 (17%)

RII = 10 (21%)

RIII = 2 (4%)

42%

3. Giboda et al., 1992 (Southeast Asian J Trop Med Pub Hlth 23: 383-388)

-  Antimalarial drug: CQ -  Nam-Ngum dam, Vientiane (1989) -  N = 15 (10 days of follow up ?)

Results

Sensitive = 9 (60%)

RI = 2 (13%)

RII = 2 (13%)

RIII = 2 (13%)

40%

4. Pillai et al., 2001 (JID 183: 789 - 95)

-  Antimalarial drug: CQ (28 days – DOT) -  Vangvieng District, Vientiane (1998-99) -  N = 39

Results

Sensitive = 21 (54%)

RI = 5 (13%)

RII - RIII = 13 (33%)

46%

5. Guthmann et al., 2002 (Ann Trop Med Parasitol 96: 553 -7)

-  Antimalarial drug: CQ (28 days – DOT) -  Sekong Province (1999 - 2000) -  N = 88

Results

ARC = 53 (60%)

ETF = 6 (7%) LTF = 29 (33%)

40%

CMPE - LAOS

Centre of Malariology,

Parasitology & Entomology

(CMPE), Ministry of

Health, Laos

Station of Malariology,

Parasitology & Entomology, Savannakhet Provincial

Health, Laos

FEUANG

XEPON PHALANXAY

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Log drug concentrations 1 2 3 4 5 6 7 8 ng/ml

EC 50

Emax

Eff

ects

(par

asite

kill

ing)

In vitro antimalarial susceptibility patterns of P. falciparum malaria in Laos

(Phanlanxay District, Savannakhet 2003 - 2004)

P. falciparum isolates defined as resistant (N = 108):

* Chloroquine 65 %

* Quinine 40 %

* Mefloquine 08 %

(Mayxay et al., 2007: Am J Trop Med Hyg 76:245-50)

-  Blood spots collected on filter papers

-  DNA extraction

-  PCR to study gene

mutation

Normal gene (DHFR)

Mutation of gene (DHFR)

Enzyme structure Target of drug

Change of enzyme structure

Serine

Asparagines

108

108

Target of drug

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Distribution of P. falciparum molecular markers associated with SP & CQ in Laos

Collaboration with Dr. Tim Anderson, Texas, USA

* Countrywide survey

* ~884 blood spot samples on

filter paper from 17 provinces

* Molecular markers:

PfDHFR & DHPS, PfCRT

Phongsaly n = 2

Luang Namtha n = 23

Bokeo n = 12

Xaya- buly

n = 6

Udomxai n = 12 Luang

Phabang n = 36

Huaphan n = 0

Xiengkhuang n = 4

Special zone n = 1

Vientiane n = 27

Vientiane Capital (no malaria transmission)

Borikhamxai n = 11

Savannakhet n = 392

Khammuan n = 16

Saravan n = 33

Champasack

n = 33

Xekong n = 28

Attapeu n = 24

No resistance mutations

1 mutation

2 mutations

3 mutations

4 mutations

Dhfr gene mutation in parasites (resistant to P)

from 17 provinces of Laos

(Mayxay et al., 2007:

Am J Trop Med Hyg 77: 36 - 43)

Phongsaly n = 2

Luang Namtha n = 23

Bokeo n = 12

Xaya- buly

n = 7

Udomxai n = 15 Luang

Phabang n = 35

Huaphan n = 0

Xiengkhuang n = 4

Special zone n = 1

Vientiane n = 29

Vientiane Capital (no malaria transmission)

Borikhamxai n = 10

Savannakhet n = 416

Khammuan n = 21

Saravan n = 34

Champasack

n = 31

Xekong n = 27

Attapeu n = 18

(Mayxay et al., 2007:

Am J Trop Med Hyg 77: 36 - 43)

No resistance mutations

1 mutation

2 mutations

3 mutations

4 mutations

Dhps gene mutation in parasites (resistant to S)

from 17 provinces of Laos Phongsaly n = 2

Luang Namtha n = 53

Bokeo n = 14

Xaya- buly

n = 7

Udomxai n = 23 Luang

Phabang n = 39

Huaphan n = 3

Xiengkhuang n = 7

Special zone n = 1

Vientiane n = 47

Vientiane Capital (no malaria transmission)

Borikhamxai n = 12

Savannakhet n = 452

Khammuan n = 25

Saravan n = 49

Champasack

n = 41

Xekong n = 36

Attapeu n = 25

76-K (Wild type)

76-T (Mutant type)

pfCRT 76-allele frequency in parasites (resistant to CQ) from 17

provinces of Laos

(Mayxay et al., 2007:

Am J Trop Med Hyg 77: 36 - 43)

Distribution of P. falciparum molecular markers associated with SP & CQ resistance in Laos

•  Countrywide (884 blood spot samples on from 17 provinces)

•  Molecular markers: pfdhfr & pfdhps; pfcrt

•  Proportion of mutation:

* Pf-dhfr (resistant to pyrimethamine): 77% with >1 mutations (164 frequency = 6%)

* Pf-dhps (resistant to sulphadoxine): 21% with >1 mutations

* Pf-crt (resistant to chloroquine): 85% with 76T mutations

(Mayxay et al., 2007. Am J Trop Med Hyg 77: 36–43)

-  Patients take antimalarial drugs -  Follow up (7, 14, 28, 42, 63… days) -  Assessment: * Cure rate * Fever clearance time * Parasite clearance time

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(Schwobel et al., 2001. Trop Med Intl Health 8: 19-24)

Attapeu Province (14 day-follow up) N = 107

Results CQ SP CQ+SP MQ (n=29) (n=28) (n=24) (n=26)

ACR 16 (55%) 23 (82%) 20 (83%) 26 (100%)

ETF 3 (10%) 1(4%) 1(4%) 0

LTF 10 (35%) 4 (14%) 3 (13%) 0 45% 18% 17%

Therapeutic Efficacy of Chloroquine plus Sulphadoxine/Pyrimethamine Compared with Monotherapy with Either Chloroquine or Sulphadoxine/ Pyrimethamine in Uncomplicated Falciparum Malaria in Laos

Follow up of patient at home

3 Days COMPARISON OF PARASITE CLEARANCE TIMES

2

1

0 CQ + SP AM AL

PCT

- Mea

n (S

D)

*

* Significant difference compared to other two groups P < 0.001

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50 Hours

* Significant difference compared to other two groups P < 0.001

COMPARISON OF FEVER CLEARANCE TIMES

40

20

30

10

0 CQ + SP AM AL

FCT

- Mea

n (S

D)

* 30 % GAMETOCYTAEMIA AFTER TREATMENT

20

10

0 CQ + SP AM AL

28/110 (25.5%)

4/110 (3.6%) 5/110 (4.5%)

*

* Significant difference compared to the other two groups (P < 0.001)

AM CQ+SP AL

*

*

*

%

* Significant difference from other groups

Neuro-psychiatric

PROBABLE SIDE EFFECTS AFTER TREATMENT

(Mayxay et al., 2006: Trop Med Intl Health 11: 1157-65 )

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3 Days

COMPARISON OF PARASITE CLEARANCE TIMES

2

1

0 AM DP

PCT

- Mea

n (S

D)

P = 0.14

30

Hours

P = 0.85

COMPARISON OF FEVER CLEARANCE TIMES

20

10

0

AM DP

FCT

- Mea

n (S

D)

Checking body temperature

PROBABLE SIDE EFFECTS AFTER TREATMENT

AM AK

%

* Significant difference between groups (P < 0.05)

*

*

*

*

*

*

* But the problem is…

Chinese GMP vs ICH GMP

A Phase III, randomized, non-inferiority trial, to assess the

efficacy and safety of Dihydroartemisinin+Piperaquine (DHA

+PPQ, Artekin) in comparison with Artesunate+Mefloquine

(AS+MQ) in patients affected by acute, uncomplicated

Plasmodium falciparum malaria in Asia

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Treatment outcome (63 days follow up)

* Median (range)

(Mayxay et al., in preparation)

Side Effects After Treatment

•  % patients with at least one recorded potential side effect was significantly higher in AM [43/98 (44%)] compared to DP [64/202 (32%)] groups (P = 0.039).

•  Incidence of post treatment dizziness, nausea, insomnia, and anorexia were all significantly higher in AM compared to DP recipients (P < 0.01).

CONCLUSION: DP is not inferior to AM in the treatment of uncomplicated falciparum malaria in southern Laos and was associated with fewer adverse effects.

Conclusions 1.  Chloroquine & sulphadoxine-pyrimethamine are not

longer efficacious for the treatment of uncomplicated falciparum malaria in Laos

2.  Artemisinin combination therapy (artesunate + mefloquine, artemether –lumefantrine or coartem, dihydroartemisinin-piperaquine or artekin): work very well in Laos

STUDY TEAM Mayfong Mayxay Samlane Phompida Siamphai Keola

Paul N Newton Rattanaxay Phetsouvanh Ratsuda Yapom Nicholas J White Maniphone Khanthavong Anna Annerberg Alan Brockman Tiengkham Pongvongsa Kasia Stepniewska Niklas Lindegardh Vonthalome Thongpaseuth Shalini Nair Marion Barends Bouakham Vannachone Tim Anderson

ACKNOWLEDGEMENTS Vanphanom Sychareun Souksavanh Bouachanh Syvoraphanh

Stephane Proux Francois Nosten Kai-Amphon Keopasert

Pitta Odai Xaisithideth Chanthala Vilaihong

Phomma Manisack Phommasansack Bounpon

Bangthon Thonglien Singnort Vilayphone

Minit Bounmy ……. Wellcome Trust of Great Britain

Lao children after malaria treatment