Mahidol-Oxford Tropical Medicine Research Unit (MORU)

1979 – 2007

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MORU - Aim

To develop effective, practical means of diagnosing and treating tropical infections responsible for significant morbidity and mortality in the populous rural areas of Southeast Asia and elsewhere.

Strategic position….

Half of the world’s population within 2000 miles….

Methods1. Clinical epidemiology2. Pathophysiology of the disease and

pathobiology of the pathogen3. Diagnosis - developing and testing new

tests4. Pharmacokinetic and pharmacodynamic

studies 5. Clinical trials6. Dissemination and translation

Paradigm of large, multi-centre clinical trials

• Severe malaria– SEAQUAMAT– AQUAMAT

• Melioidosis– Thailand Melioidosis Clinical Trials Group– MERTH– ATOM

• Scrub typhus– Vientiane + Udon Thani

• Haemofiltration vs Haemodialysis (planned)– India + Viet Nam

Hub and Spoke topology…

Major field site / laboratory

Laos project field site

Bangkok Unit

Collaborator’s site

MozambiqueAQUAMAT collaborative sitesSri Lanka

MORU - 2007

Collaborative siteClinical study site

‘Thailand’ Unit - Clinical Study Sites

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MORU - Staff• 210 host country staff• 27 international staff

– 8 British– 4 Dutch– 3 Swedish– 2 Americans– 2 Australians– 2 French– 1 Swiss– 1 Polish– 1 Japanese– 1 Singaporean– 1 Burmese– 1 Canadian

DiseasesMalariaMelioidosisRickettsial illnessesStaphylococcal infectionsLeptospirosisCryptococcal meningitisBeriberiDengueTyphoidAcute respiratory infections

Unit Publications

Melioidosis

Malaria

Snake bite

Other micro

Other

1981 - 2004 2005-2006

- Uncomplicatedmalaria

- Severe malaria- Malaria in

pregnancy

SMRUSEAQUAMAT

Current treatment recommendations for

Treatment of severe malaria

• Can we lower the mortality of severe malaria by using artesunate rather than quinine?

IV Artesunate vs IV Quinine multi-centre trial in Asia - ‘SEAQUAMAT’

SEAQUAMAT Study sites

Overall 35% (19% to 48%) reduction in mortality with artesunate

SEAQUAMAT

‘Numbers needed to treat’ (NNT)

• Burma 12.6 (7.3 to 45)• Bangladesh 11.1 (5.8 to 121)• India 21.2• Indonesia 16.6

SEAQUAMAT

‘Artesunate is the recommended first choice in areas of low malaria transmission’

‘In pregnant women…. artsunate is the first and artemether the second option in the 2nd and 3rd trimesters’

‘In the first trimester, until more evidence becomes available, both artesunate and quinine may be considered as options’

AQUAMAT

• Artesunate vs quinine in African children with severe malaria

• Powered to detect a 25% reduction in mortality from 8% to 6%

• Sample size: 5,306• Aiming for 12 sites in 8 countries• Funded by the Wellcome Trust

AQUAMAT

• Mozambique - 1 sites• Kenya - 2 sites• The Gambia - 1 sites• Ghana - 1 site• Tanzania - 3 sites• Uganda - 1 site• Rwanda - 2 sites• Nigeria - 2 sites

AQUAMAT - Current status• Sites actively recruiting now

– Mozambique - Beira– Ghana - Kumasi – Kenya - Kilifi– The Gambia - Banjul– Tanzania - Muheza & Korogwe

• 1,236 cases recruited as of 6/9/07• 123 deaths (10%)

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AQUAMAT - Current status• Sites actively recruiting now

– Mozambique - Beira– Ghana - Kumasi – Kenya - Kilifi– The Gambia - Banjul– Tanzania - Muheza & Korogwe

• 1,331 cases recruited as of 1/10/07• 133 deaths (10%)

Levamisole in severe malaria

• Chittagong Medical College• Pilot study• Outcome markers:

– Serial parasite staging– Cytoscan– Lactate clearance

• 30 patients randomised so far…

Fake antimalarials

Drug resistance

Developments 2006-2007Artemisinin resistance?

• West Africa - in vitro evidence??• Cambodia

– prolonged PCT– Currently under investigation

• Artemisinin resistance would be a colossal disaster…

Pailin, SW Cambodia

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Pharmacology

The new pharmacology laboratory January 2007

PharmacologyDevelopments 2006-2007

• Development of new assays for:– Amoxycillin– Piperaquine

• Plasma• Blood spots• Urine• High throughput

– Lumefantrine - high throughput– Oseltamivir– Zanamivir– Artesunate / DHA

• Piperaquine– Identification of metabolites– Identification of major manufacturing impurity

• New method for assessing regression models for standard curves

PK studies in disease• In pregnancy

– Amodiaquine– Arthemether-lumefantrine (levels reduced in pregnancy)– Artesunate

• In uncomplicated malaria– Artesunate (Pailin)– Lumefantrine (OD dosing inadequate)– Piperaquine (OD dosing OK)

• In melioidosis– Co-amoxiclav

• In avian and severe seasonal influenza (SEA001)– Oseltamivir

Effect of loading dose – 1.5x (red), 2x (green) maintenance. Dose = 75mg / 12h.Effect of probenecid (orange). Dose = 75mg /24h. Ka different for the first dose.

OC concentration (ng/ml)

Time (h)

Largest and most detailed pharmacokinetic evaluation of oseltamivir;4 different doses, loading dose, probenecid ~1800 samples

Microbiology

Fever studies

• Completed– Vientiane– Udon Thani– Nepal

• Ongoing– Thai-Burmese border– Chiang Rai– Siem Riep– Colombo

Fever studies - major findings

• Leptospirosis - common in rural areas• Rickettsial diseases

– Scrub typhus– Murine typhus

• Dengue and Chikungunya• Main blood culture pathogens variable

– Vientiane & Nepal - typhoid– NE Thailand - melioidosis

Diagnostics• Need to be tailored to the region - targeted by fever

study• Robust, accurate, cheap• Blood culture

– Conventional and specialised (rickettsial, leptospire)• Dipsticks - Ag and Ab detection• PCR

– Malaria, scrub and murine typhus, dengue, melioidosis, leptospirosis

– Conventional, real time, LAMP• Fluorescence - Ag and Ab detection

– Melioidosis, scrub and murine typhus

Dengue rapid tests - overall diagnostic performance

n Core Diazyme Globalemed Panbio SD Teco Tulip Minerva

Sn 501 22.9

(18.3-27.6)

17.8

(13.8-22.4)

62.9

(57.4-68.1)

65.3

(59.9-70.5)

21.8

(17.4-26.7)

9.5

(6.6-13.2)

6.4

(4.0-9.7)

8.6

(5.8-12.2)

Sp 501 98.9

(95.9-99.9)

97.7

94.3-99.4)

68.0 (60.5-74.8)

96.6

(92.7-98.7)

98.3

(95.1-98.7)

95.4

(91.2-98.0)

99.4

(96.9-99.9)

100

(97.9-100)

PPV 501 97.4

(90.8-99.7)

93.6

(84.3-98.2)

78.5(73.1-83.4)

97.3

(94.1-99.0)

96.0

(88.6-99.2)

79.5

(63.5-90.7)

95.5

(77.2-99.9)

100

(87.7-100)

NPV 501 40.7

(36.0-45.6)

39.0

(34.4-43.7)

49.6

(43.1-56.1)

59.9

(54.0-65.7)

40.3

(35.6-45.1)

36.2

(31.8-40.7)

36.3

(32.0-40.8)

37.0

(32.6-41.5)

Tsutsugamushi disease(scrub typhus)

– Acute febrile illness

– Causative agent- Orientia tsutsugamushi

– Transmitted to humans by bite of larval stage of Leptotrombidium spp. mite (chigger)

– Well suited to antibiotic treatment

– Untreated mortality 2% to 36%

– No vaccine

Mae sot

Bangkok

Udon Thani

565 km512 km

O. tsutsugamushi culture- transport from clinical sites

Results - in vitro isolationUdon Thani (UT) Mae Sot (FPW)

Patient samples( n=41 )

28 13

3

Median: 4.0*Range: 2-7

Median: 22**Range: 22-25

Isolates( n=17 )

Positivity rate = 41.5%

14

Transit time (days) Median: 2.0*Range:1-5

Time to isolation (days) Median: 22** Range: 7-37

Luksameetanasan et al., 2006 - Monday’s poster session

O. tsutsugamushi MLST

Determining major infectious pathogens affecting rural populations• Fever studies• Development of cheap diagnostics

– e.g. ‘fever stick’• Rickettsial diseases, leptospirosis, dengue• Malaria antigen detection• CRP

• Trials to determine appropriate empirical therapy

Leptospirosis in Udon Thani

0.00

0.01

0.02

0.03

ST-34

L. kirschneri

ST-17ST-22

ST-27ST-41

ST-29

ST-37

ST-46

ST-49

A

Udon ThaniOther ThaiRodent isolate

Reference strain

G-CSF for severe melioidosis • Randomized, placebo controlled trial, was conducted to assess the

efficacy of lenograstim (G-CSF 263 mcg iv daily) in patients with severe sepsis due to suspected melioidosis in Ubon Ratchathani, Thailand, during 2004-2006.

• 60 patients were enrolled

• Mortality was similar in both groups (G-CSF 70% vs placebo 87%, p=0.2) including 41 patients with confirmed melioidosis (83% vs 96%, p=0.3)

Hazard ratio 0.56 (95%CI 0.31-1.00, p=0.05)

MERTH : Melioidosis ERadication THerapy/THailand

Doxycycline plus Trimethoprim/sulfamethoxazole

versus Trimethoprim/sulfamethoxazole

as Maintenance Therapy for Melioidosis Prospective, multicentre, randomised, double-blind, placebo-control trial

KKU, KK, Ubon, Udon and MK Sample size 635 cases. 270 enrolled by end

Aug

ATOM: RCT of Ceftazidime versus Meropenem as Parenteral

Therapy for Severe Melioidosis

Septic patients with suspected melioidosis requiring parenteral antibiotics.

Prospective, multicentre, double-blind, randomisedcontrol trial

Ubon and UdonSeptember 07 – September 10Sample size 650

Uncomplicated malaria

Leptospirosis

Severe malaria Melioidosis

Scrub typhus Murine typhus

n=82,616

n=150

n=6,636

n=335n=874

n=1,357

All clinical trials - patients randomised by MORU as percentage of all patients randomised worldwide since

1960

Long term investment in people and infrastructure…

• Continuous core-funding from the Wellcome Trust

• MORU - 3 directors in 28 years• OUCRU, VN - 2 directors in 16 years

Current publication output…

• One every 4.6 days• Including OUCRU, VN, one every 2.3

days

0

10

20

30

40

50

60

70

80

90

19811982198319841985198619871988198919901991199219931994199519961997199819992000200120022003200420052006

Year

Solid line - all publications (total 832)Dotted line - peer reviewed original articles in international journals (total 633)

MORU - Publications 1981 to 2006

Acknowledgements

• Unit Staff in Thailand and Laos

• SEAQUAMAT, AQUAMAT & other collaborators

• Mahidol & Oxford Universities

• NIH

• The Wellcome Trust

Effect of N-acetylcysteine on lactate clearance in severe malaria

Watt et al, QJM 2002

NAC as an adjunct to artesunate in severe malaria

(n=108)

NAC and coma clearance

Major field site / laboratory

Bangkok Unit

Thailand Unit - 1979

Major field site / laboratory

Bangkok Unit

Thailand Unit - 1979

1. Rediscovery of quinidine as an antimalarial.2. Pharmacological basis for quinine and quinidine dosing in

uncomplicated and severe malaria (1) (became USA treatment of choice for severe malaria).

3. Introduction of quinine loading dose in severe malaria (1). (Endorsed by WHO since 1986)

4. Discovery of quinine induced hyperinsulinaemia (2). (The major adverse effect of quinine in malaria).

5. Pharmacological basis for chloroquine dosing in uncomplicated and severe malaria (then the most widely used treatment in the world).

6. Introduction of revised chloroquine regimens in severe malaria (3). Endorsed by WHO.

Malaria Antimalarial drug research

1980-1989

7. The evaluation and introduction of the artemisinin derivatives; (Now accepted as the best antimalarial drugs. Artemisinin based combination treatment {ACT} has become internationally recommended first line treatment)8. First study of an artemisinin derivative in Africa9. A series of large community based studies leading to wide-scale introduction of ACTs.10.Demonstration that artemisinin derivatives reduce gametocyte carriage (transmissibility), provides rationale for their use in malaria control.11. Discovery of halofantrine cardiotoxicity (led to the drug being generally abandoned).

MalariaAntimalarial drug research

1990-1999

MalariaAntimalarial drug research

1990-1999

12. Evaluation and introduction of high dose split dose mefloquine (now endorsed by WHO as the correct method of administration). 13. Development of a model for antimalarial pharmacodynamics and introduction of the population mixed-effects modelling approach to the evaluation of antimalarial drugs.14. Development of genotyping methods for field studies of falciparum malaria (allowed large community based studies to be conducted for the first time as reinfection could be distinguished from recrudescence)14. Pharmacokinetic-pharmacodynamic studies of artemether-Lumefantrine (lead to a new dose regimen; now the most widely used ACT in the world)

16. Conducting the largest ever series of antimalarial drug studies over a ten year period showed that systematic deployment of ACTs resulted in sustained cure rates over 90%, reduced the incidence of malaria over ninefold, and led to a reversal of mefloquine resistance. (These studies provided a justification for the largest ever trials in Africa, and the WHO recommendation in 2001 that any country changing antimalarial drug policy should switch to ACTs. WHO guidelines now recommend ACTs as first-line everywhere)17. First melding of pharmacokinetic and pharmacodynamic models to examine the propensity for antimalarial drug resistance to develop. This provided a solid rationale for never deploying initially a low dose of mefloquine – as had been done in SE Asia. This work was based on a novel description of in-vivo antimalarial pharmacodynamics which is now generally accepted and used as a basis for deriving antimalarial drug regimens.

Malaria Antimalarial drug research

1990-1999

18. Description and analysis of the factors which lead to the emergence of antimalarial drug resistance. This built on models developed over eight years. This identified inadequately treated hyperparasitaemic patients as the main source of de-novo resistance, and not recipients of prophylaxis or exposure to repeated new infections during the elimination phase of an antimalarial drug (as had been generally thought). This and associated work has been very influential in the development of strategies for global antimalarial drug deployment19. Introduction of new methods of assessing falciparum and vivax malaria (now endorsed by WHO)20. Introduction and dose optimisation of a new ACT; dihydroartemisinin-piperaquine21. Conduct of the largest ever trial in severe malaria which showed that artesunate reduced the mortality of severe malaria by 35% compared with quinine (immediately adopted as recommendation by WHO)

Malaria Antimalarial drug research

2000-2007

1. First studies of cerebral blood flow and metabolism in cerebral malaria

2. Description of hypoglycaemia and acidosis as major pathological processes in severe malaria .

3. Discovery that the entire red cell population in severe malaria (but not sepsis) becomes less deformable and that this is a major determinant of outcome (subsequently found to result from haem liberation at schizogony).

4. First quantitative studies of sequestration in fatal falciparum malaria shows that cerebral sequestration occurs in all patients, but is more in cerebral malaria, and that the process is highly variable between blood vessels, that once parasitized erythrocytes adhere they do not subsequently detach, and that ring stages also sequester.

MalariaPathophysiology

5. Characterisation of cytoadherence and rosetting as pathogenic factors in human malarias.

6. Identification of the first clear virulence determinant in Plasmodium falciparum (a failure of density dependent inhibition of multiplication).

7. Fever was shown to accelerate the cytoadherence of Plasmodium falciparum. This had important practical consequences as earlier studies had misinterpreted the slower decline in parasitaemia associated with antipyretics as harmful and recommended against use of these drugs. This study suggested the opposite and provided a clear justification for the use of paracetamol (which was the most widely used drug treatment of malaria apart from antimalarials in the world!)

MalariaPathophysiology

8. Largest prospective studies of malaria in pregnancy in a low transmission setting (showed the value of regular antenatal clinics in reducing mortality)

9. Demonstration that vivax malaria reduces birthweight (major implications for control programmes)

10. Largest prospective studies with artemisinin derivatives in pregnancy (central to current WHO recommendations)

MalariaPathophysiology

5. Characterisation of cytoadherence and rosetting as pathogenic factors in human malarias.

6. Identification of the first clear virulence determinant in Plasmodium falciparum (a failure of density dependent inhibition of multiplication).

7. Fever was shown to accelerate the cytoadherence of Plasmodium falciparum. This had important practical consequences as earlier studies hadmisinterpreted the slower decline in parasitaemia associated with antipyretics as harmful and recommended against use of these drugs. This study suggested the opposite and provided a clear justification for the use of paracetamol (which was the most widely used drug treatment of malaria apart from antimalarials in the world!)

MalariaPathophysiology

Can didat e inter ve ntio n Sc ien t if ic rati onal e

Evide nc e so f a r

Prac t ic a l ity Cont rove rsy Sta t us

Parente ral a r t esunat e vs quinine i n Afri can children

***** ***** ***** ** Under way

Aggre ssive fl uid res uscit at ion in c hildren

*** *** *** ***** Funded

Levamisole **** *** **** ** Pilot

Excha nge blood tra nsf usion ***** *** * *** No p lans

Empirical a nti biot ics in high tra nsmission ar eas

***** **** ***** **** No p lans

Dichloroace ta te **** *** ** **** No p lans

Ear ly haemof ilt ra t ion *** **** * *** No p lans

N- ac ety lcyst eine **** *** ** ** No p lans

Venti la t ion an d s edat ion ***** **** * *** No p lans

Mannit ol in cer ebral malar ia

** * *** **** No p lans

Opt imal fl uid ma nagem ent in adu lt s

**** ** *** *** Pilot planned

Feeding st ra teg ies **** * **** *** No p lans

Large severe malaria trial intervention candidates

Thailand 2003, Newton et al CID, Vietnam 1997, Cao et al, Trans R Soc Trop Med HygVietnam 1992, Hien et al, Trans R Soc Trop Med Hyg Myanmar 1992, Win et al, Trans R Soc Trop Med Hyg

All randomised trials

SEAQUAMAT study

Subgroup analysis - Age

Odds ratio.194486 .5 1 1.5

Combined

ADULT

CHILD

AQUAMAT - planned new sites

• Uganda - Mbarara• Tanzania - Tanga• Kenya - Mombasa• Nigeria - Ilorin, Jos• Rwanda - Mbilisi, Rwamagana

• Aim to finish by March 2010

Determining major infectious pathogens affecting rural populations

• Fever studies to determine clinical epidemiology• Development of affordable diagnostics

– Blood spots– Molecular - PCR, Real time-PCR, lamp– Dipsticks

• Trials to determine appropriate empirical therapy– Doxycycline vs azithromycin– SUT and MUT

UT169

UT76

0.02

UT167UT316

UT150

UT177UT176

UT219UT213UT221

FWP2031

UT302FWP2016

FWP1038

UT196UT144

UT125

Udorn Thani

Mae Sot

Genetic distance relative to geographical origin of Thai O. tsutsugamushi isolates

KARP

GILLIAM TA763

TA716

Adjuvant therapies in severe malaria

• None clinically proven so far• Trials generally too small or poor quality

Levamisole

• Levamisole appears to inhibit sequestration of trophozoites in vivo

• Potential adjunctive therapy for severe malaria

• Trial as adjunct to artesunate in severe malaria

Yipp et al. Blood 2003; 101: 2850-2857

1. IRBC binds to CD36

2. This activates the associated SRC-family kinases

3. Which activates an Ecto-alkaline phosphatase

4. Dephosphorylation of CD36 sharply increases the binding affinity of CD36 for IRBCs.

Yipp et al. Blood 2003; 101: 2850-2857

Levamisole is a widely used anti-helminthic drug which specifically inhibits cell-bound ALP.

Levamisole (500μM) inhibits cytoadhesion by 50%

(HDMEC monolayer and SCID mouse model)

LEVAMISOLE

JID 2007, 196:460

Major field site / laboratory

Bangkok Unit

Viet Nam Unit

MORU - 2000

MORU contributions to SEAICRN

• Volunteer studies– Oseltamivir (SEA002)

• Doses up to 450mg• Effect of loading dose• Effect of probenecid

– IV zanamivir / oral oseltamivir interaction study (SEA003) - imminent– Long term prophylaxis study (SEA004) - planned

• N=600. Zanamivir (inhaled) vs oseltamivir (po)• 16 weeks - safety and tolerability• ??Neuropsychiatric side effects

• Pharmacology - drug measurement and analysis