MIM/TDR ANTIMALARIAL DRUG RESISTANT MIM/TDR ANTIMALARIAL DRUG RESISTANT NETWORK IN NIGERIA NETWORK IN NIGERIA : : Defining and monitoring drug resistant Plasmodium falciparum infections in south west Nigeria Investigator’s Name Investigator’s Name : Dr. Grace Olusola Gbotosho : Dr. Grace Olusola Gbotosho Institution: Malaria Research Laboratories, Institution: Malaria Research Laboratories, Postgraduate Institute for Medical Research and Postgraduate Institute for Medical Research and Training, College of Medicine, University of Training, College of Medicine, University of Ibadan. Ibadan.
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MIM/TDR ANTIMALARIAL DRUG RESISTANT MIM/TDR ANTIMALARIAL DRUG RESISTANT NETWORK IN NIGERIANETWORK IN NIGERIA: : Defining and monitoring drug resistant Plasmodium falciparum infections in south west Nigeria
Investigator’s NameInvestigator’s Name: Dr. Grace Olusola Gbotosho: Dr. Grace Olusola Gbotosho
Institution: Malaria Research Laboratories, Postgraduate Institute for Institution: Malaria Research Laboratories, Postgraduate Institute for Medical Research and Training, College of Medicine, University of Medical Research and Training, College of Medicine, University of Ibadan.Ibadan.
OBJECTIVOBJECTIVESES
SCIENTIFIC OBJECTIVESSCIENTIFIC OBJECTIVES::• Definition of the characteristics of Definition of the characteristics of P. falciparumP. falciparum to chloroquine (CQ), to chloroquine (CQ),
• Collation of clinical, parasitological (in vitro & molecular) and Collation of clinical, parasitological (in vitro & molecular) and pharmacokinetic data following treatment with Chloroquine or pharmacokinetic data following treatment with Chloroquine or sulphadoxine-pyrimethamine.sulphadoxine-pyrimethamine.
• Determination of current levels of resistance to chloroquine, sulphadoxine-Determination of current levels of resistance to chloroquine, sulphadoxine-pyrimethamine.pyrimethamine.
• Strengthening the capacity to address the problem of drug resistant malaria Strengthening the capacity to address the problem of drug resistant malaria in Nigeria using targeted research as a vehicle for capacity building.in Nigeria using targeted research as a vehicle for capacity building.
CAPACITY BUILDING OBJECTIVESCAPACITY BUILDING OBJECTIVES Establish capacity sustainable to implement the 5 main aspects of the Establish capacity sustainable to implement the 5 main aspects of the
network protocol (Clinical, network protocol (Clinical, in vitroin vitro, molecular, drug analysis and data , molecular, drug analysis and data management)management)
Train health care providers in diagnosis and management of drug Train health care providers in diagnosis and management of drug resistant malaria.resistant malaria.
Provide local data to support malaria control policy.Provide local data to support malaria control policy.
Improve research infrastructure at the institutionImprove research infrastructure at the institution
PARTNERS AND COLLABORATING PARTNERS AND COLLABORATING INSTITUTIONSINSTITUTIONS
Walter Reed Army Institute of Research, USA Walter Reed Army Institute of Research, USA (Dr. Dennis Kyle) (Dr. Dennis Kyle)
Harvard School of Public Health, USA Harvard School of Public Health, USA (Prof. Dyann Wirth) (Prof. Dyann Wirth) Thammasat University Thailand Thammasat University Thailand (Dr. Kesara Nabachang) (Dr. Kesara Nabachang)
Karolinska InstituteKarolinska Institute ( (in discussionin discussion))
BACKGROUND AND RATIONALE FOR BACKGROUND AND RATIONALE FOR PROJECTPROJECT
Chloroquine resistant Chloroquine resistant P. falciparumP. falciparum infections represent infections represent a major public health problem in Nigeria.a major public health problem in Nigeria.
In most areas where chloroquine resistance has In most areas where chloroquine resistance has emerged, treatment of malaria relies on the use of emerged, treatment of malaria relies on the use of alternative antimalarial drugs including:alternative antimalarial drugs including:
Prolonging the clinical life of alternative antimalarial drugs:• Promotion of rational drug use • Improved treatment protocols
Monitoring the spread of drug resistant malaria using standard protocols.
Documentation of drug resistant malaria.
Challenges of Antimalarial Drug Resistance in Africa:
This study addressesThis study addresses:: Monitoring the spread of drug resistant malaria using
standard protocols. Documentation of drug resistant malaria.
Antimalarial drug resistance is being evaluated based on:Antimalarial drug resistance is being evaluated based on:
• Clinical outcome of treatment in the patientClinical outcome of treatment in the patient• In vitro drug susceptibility profile of the parasiteIn vitro drug susceptibility profile of the parasite• molecular markers of drug resistance in the parasitemolecular markers of drug resistance in the parasite• blood level of the antimalarial drug in the patientblood level of the antimalarial drug in the patient
This study is providing an opportunity for understanding the distribution This study is providing an opportunity for understanding the distribution
and level of chloroquine resistant malaria in Nigeria in a systematic wayand level of chloroquine resistant malaria in Nigeria in a systematic way ..
STUDY DESIGN AND METHODOLOGYSTUDY DESIGN AND METHODOLOGY Study sites:Study sites:
• A rural site (Olode-Adetoun and Gbedun villages)A rural site (Olode-Adetoun and Gbedun villages)• Urban site (Adeoyo Maternity Hospital and the Malaria Clinic, UCH)Urban site (Adeoyo Maternity Hospital and the Malaria Clinic, UCH)
Sample size: 300Sample size: 300
Clinical studiesClinical studies::• Study received ethical approval from the joint UI/UCH ethical Study received ethical approval from the joint UI/UCH ethical
review committee.review committee.
• Drug efficacy studies were initiated at the urban study sites.Drug efficacy studies were initiated at the urban study sites.
• Patients aged 6months to 5 years with clinical symptoms Patients aged 6months to 5 years with clinical symptoms microscopically confirmed microscopically confirmed P. falciparumP. falciparum infection and were infection and were enrolled after informed consents from parent/guardian.enrolled after informed consents from parent/guardian.
• Blood was obtained from each patient for hematology and biochemistry prior to Blood was obtained from each patient for hematology and biochemistry prior to treatment and selected time intervals.treatment and selected time intervals.
• Patients were treated with standard doses of either chloroquine or sulphadoxine-Patients were treated with standard doses of either chloroquine or sulphadoxine-pyrimethamine.pyrimethamine.
• Each patient was followed up for 14 days to monitor clinical response to treatment.Each patient was followed up for 14 days to monitor clinical response to treatment.
Parasite susceptibility and laboratory studiesParasite susceptibility and laboratory studies
• Venous blood was obtained from each patient prior to treatment Venous blood was obtained from each patient prior to treatment and at recrudescence of infection for in vitro assayand at recrudescence of infection for in vitro assay
• Susceptibility of patient isolates to standard antimalarial drugs Susceptibility of patient isolates to standard antimalarial drugs was performed using:was performed using:
– A modification of the standard WHO schizont inhibition assay A modification of the standard WHO schizont inhibition assay based on the incorporation of selected resistance reversing agents based on the incorporation of selected resistance reversing agents (verapamil). (verapamil).
– The double Elisa assay (DELI) based on pLDH activity The double Elisa assay (DELI) based on pLDH activity
Molecular Assays:Molecular Assays:• Blood samples from each patient were blotted on filter paper Blood samples from each patient were blotted on filter paper
prior to treatment, during follow up and at recrudescence. prior to treatment, during follow up and at recrudescence.
• Filter papers were dried at room temperature and stored until Filter papers were dried at room temperature and stored until used for DNA extractionused for DNA extraction. .
• Nested PCR AND RLFP analysis were used to evaluate the Nested PCR AND RLFP analysis were used to evaluate the prevalence of mutations in the pfcrt and pfmdr1 genes of prevalence of mutations in the pfcrt and pfmdr1 genes of isolates of P. falciparum obtained from patients before isolates of P. falciparum obtained from patients before treatment and during recrudescence of infection. treatment and during recrudescence of infection.
• Genotypes of Genotypes of Plasmodium falciparumPlasmodium falciparum population, were population, were determined using polymorphic markers (MSP-1, MSP-2 and determined using polymorphic markers (MSP-1, MSP-2 and GLURP) prior to treatment and during recrudescence from GLURP) prior to treatment and during recrudescence from filter paper samples.filter paper samples.
Antimalarial drug blood levelsAntimalarial drug blood levels• 100ul blood samples were blotted on filter paper prior to 100ul blood samples were blotted on filter paper prior to
treatment and on days 1, 3, 7 and 14 after treatment and at treatment and on days 1, 3, 7 and 14 after treatment and at recrudescence, dried at room temperature and stored until recrudescence, dried at room temperature and stored until HPLC analysis.HPLC analysis.
• 1 ml Venous blood samples were obtained from patients 1 ml Venous blood samples were obtained from patients treated with sulphadoxine pyrimethamine and the red treated with sulphadoxine pyrimethamine and the red blood cells were separated from plasma and both fractions blood cells were separated from plasma and both fractions were stored till analysis. were stored till analysis.
• The primary objective of this protocol is to correlate drug The primary objective of this protocol is to correlate drug concentration with the clinical response. concentration with the clinical response.
RESULTSRESULTSClinical Outcome: Clinical Outcome: 140 patients completed 14 day follow up in the 140 patients completed 14 day follow up in the urban site. 71 patients received chloroquine while 61 patients received urban site. 71 patients received chloroquine while 61 patients received sulphadoxine-pyrimethamine. sulphadoxine-pyrimethamine. Infection in 48% of patients treated with Infection in 48% of patients treated with CQ responded to treatment while infection in 74% of patients treated with CQ responded to treatment while infection in 74% of patients treated with SP responded to treatmentSP responded to treatment
48%
74%
14%
3%
38%
23%
0%10%20%30%40%50%60%70%80%
ACR ETF LTF
CQSP
In vitro sensitivity assays: In vitro susceptibility profile of 42 patient isolates of P. falciparum have been determined. 54% of the isolates of P. falciparum were resistant to chloroquine in vitro while 46% were sensitive.
48%52% 54%
46%
0%
20%
40%
60%
80%
100%
clinicaloutcome
in vitroprofile
in vitroprofile
CQ SensitiveCQ resistantMQ sensitiveMQ resistant
CORRELATION BETWEEN IN VIVO AND IN VITRO RESPONSE: 65% of the isolates of P. falciparum obtained from patients whose infection responded favourably respond to treatment with CQ were sensitive in vitro. 66% of isolates of P. falciparum obtained from patients whose infection failed to respond to chloroquine were resistant to chloroquine in vitro.
48%
65% 66%
52%
0%
20%
40%
60%
80%
100%
clinical outcome
in vivo/ invitrosensitivein vivo/ in vitroresistantclinical resistance
Molecular Profiles: Molecular Profiles: Collation of sequence polymorphisms Collation of sequence polymorphisms at codon 86 of the pfmdr 1 gene in pretreatment samples at codon 86 of the pfmdr 1 gene in pretreatment samples
and clinical response to chloroquine treatmentand clinical response to chloroquine treatment
Pfmdr 1 polymorphism and clinical outcome: Preliminary data (from 35 patient isolates) on correlation of pfmdr 1 Y86 mutation and clinical failure to chloroquine treatment, revealed a correlation between treatment failure and the presence of the Y86 mutation in P. falciparum genes in parasite obtained from post treatment failures.
46%
14%
0%
10%
20%
30%
40%
50%
clinical
Clinicalresistance
pretreatmentmolecularprofile
46%43%
0%
10%
20%
30%
40%
50%
clinical
Clinicalresistance
Post-treatmentmolecularprofile
Correlation between gene polymorphism (pfmdr 1) and in vitro chloroquine susceptibility: 43% of the patient isolates of P. falciparum showed in vitro resistance to chloroquine. 30% of these isolates harbored the mutant pfmdr1 Y86 while the remaining isolates showed either mixed (wild type and mutant) or wild type alone. 57% of the isolates were chloroquine resistant and 30% of these also harbored the mutant pfmdr 1 Y86 allele.
43%
30%
57%
30%
0%
10%
20%
30%
40%
50%
60%
resistant sensitive
in vitropfmdr 1 Y86
CORRELATION OF TREATMENT FAILURE , BLOOD CORRELATION OF TREATMENT FAILURE , BLOOD LEVELS AND MOLECULAR PROFILESLEVELS AND MOLECULAR PROFILES
Patient IDPatient ID Day of failureDay of failure CQ blood levelCQ blood level Pfcrt allele (Do)Pfcrt allele (Do) Pfcrt allele atPfcrt allele atRecrudescenceRecrudescence
Blood level of chloroquine in patients whose Blood level of chloroquine in patients whose infection responded to chloroquine treatmentinfection responded to chloroquine treatment
PATIENT IDPATIENT ID CHLOROQUINE BLOOD LEVEL CHLOROQUINE BLOOD LEVEL (ng/ml)(ng/ml)
Patients with treatment failure:Patients with treatment failure:
The concentration of CQ ranged from 494.6ng/ml to 2243.2ng/ml (mean CQ The concentration of CQ ranged from 494.6ng/ml to 2243.2ng/ml (mean CQ in blood was 1365.1 + 547.7ng/ml).in blood was 1365.1 + 547.7ng/ml).
The CQ concentration in patients who failed treatment was higher than in The CQ concentration in patients who failed treatment was higher than in patients with CQ sensitive infections.patients with CQ sensitive infections.
58% of the isolates obtained from patients who failed treatment had mutant 58% of the isolates obtained from patients who failed treatment had mutant pfcrt T76 allele at Do.pfcrt T76 allele at Do.
33% of the isolates had both the wild type and mutant allele of pfcrt at Do.33% of the isolates had both the wild type and mutant allele of pfcrt at Do.
At recrudesccence:At recrudesccence:• 58% of the isolate had mutant allele58% of the isolate had mutant allele• 25% had both wild and mutant alleles25% had both wild and mutant alleles
MILESTONES & TIMELINES for 2nd yearMILESTONES & TIMELINES for 2nd year Completion of microscopic evaluation and data analysis of in vitro Completion of microscopic evaluation and data analysis of in vitro
studies conducted in 2002 and initiation of analysis of 2003 studies studies conducted in 2002 and initiation of analysis of 2003 studies (January 2003 to November2003)(January 2003 to November2003)
Completion of molecular assays of all samples obtained from the Completion of molecular assays of all samples obtained from the studies conducted in 2002 and initiation of analysis of 2003 studies studies conducted in 2002 and initiation of analysis of 2003 studies (January 2003 to November 2003)(January 2003 to November 2003)
Pharmacokinetic analysis of samples collected in 2002 and initiation Pharmacokinetic analysis of samples collected in 2002 and initiation of analysis of 2003 studies of analysis of 2003 studies (January 2003 to November 2003)(January 2003 to November 2003)
Training on Analytical techniques for each network site Training on Analytical techniques for each network site (May to (May to November 2003)November 2003)
Training on Standardization and evaluation of clinical protocols Training on Standardization and evaluation of clinical protocols (April 2003)(April 2003)
Group Training on Diagnosis and clinical management Group Training on Diagnosis and clinical management of drug resistant malaria of drug resistant malaria (April 2003)(April 2003)
Patient recruitment and Clinical studies at The village Patient recruitment and Clinical studies at The village sites and the urban areas (transmission season) sites and the urban areas (transmission season) (May (May 2003 to September 2003)2003 to September 2003)
Training on safe practices and data quality assurance. Training on safe practices and data quality assurance. (June 2003)(June 2003)
Collation and analysis of clinical data Collation and analysis of clinical data (September 2003 (September 2003 to November 2003)to November 2003)
CAPACITY BUILT (Year 1)CAPACITY BUILT (Year 1)Short Training :
Trainee Subject Location
Onikepe Folarin Molecular biology Uganda
Foluso Falade Molecular biology Uganda
Oyin Oduola in vitro techniques Benin RepublicYomi Sijuade in vitro techniques Benin Republic
Ayo Bamigboye in vitro techniques Benin Republic
Sola Gbotosho data mgt/team building Scotland
Lanre Bello Data mgt/team building Scotland
CAPACITY BUILT (Year 1)CAPACITY BUILT (Year 1)Name of trainee Level Name of trainee Level Specialty Specialty DurationDuration Year of enrolment Year of enrolment
Abayomi SijuadeAbayomi Sijuade *PhD Parasite biology & *PhD Parasite biology & 4 years 4 years 20032003Analytical tech.Analytical tech.
• *Institution of training: College of Medicine, University of Ibadan*Institution of training: College of Medicine, University of Ibadan
CAPACITY BUILT AS A RESULT OF THE PROJECTCAPACITY BUILT AS A RESULT OF THE PROJECT
Specialized Training:
Dr. Christian Happi: Application of advanced molecular techniques (October 2002 to December 2002) at The Harvard School of Public Health, and Walter Reed Army Institute of Research, Washington DC.
Miss Oyin Oduola: DELI assay Techniques and its application to evaluation of drug susceptibility testing of field isolates of P. falciparum (June 2002 to August 2002) at the Walter Reed Army Institute of Research, Washington DC
ENABLING FACTORS AND CHALLENGESENABLING FACTORS AND CHALLENGESFacilitating factors:Facilitating factors:
The involvement of a network management team and other technical The involvement of a network management team and other technical consultants has been useful in starting the project. consultants has been useful in starting the project.
The workshops have been very useful especially in problem solving The workshops have been very useful especially in problem solving and introduction of the new techniques.and introduction of the new techniques.
The equipment acquired on previous MIM/TDR project provided an The equipment acquired on previous MIM/TDR project provided an enabling research environment and facilitated research activitiesenabling research environment and facilitated research activities
Financial support from the College of Medicine and facilities from Financial support from the College of Medicine and facilities from
collaborating labs in the US pending arrival of the project funds.collaborating labs in the US pending arrival of the project funds.
ENABLING FACTORS AND CHALLENGES IIENABLING FACTORS AND CHALLENGES IIManagment problems:Managment problems:
The lack of fundsThe lack of funds was a limiting factor in project was a limiting factor in project execution. This was important as large part of the execution. This was important as large part of the supplies have to be obtained from overseas.supplies have to be obtained from overseas.
Equipment repairs:Equipment repairs: The prolonged waiting periods for The prolonged waiting periods for replacement of faulty equipment parts limited some replacement of faulty equipment parts limited some aspects of the research activities especially the analysis of aspects of the research activities especially the analysis of drugs in the patients samples.drugs in the patients samples.
Lack of functional uninterruptible internet connectivityLack of functional uninterruptible internet connectivity also confounded some aspects of the research effort as also confounded some aspects of the research effort as contact with other investigators on the network was contact with other investigators on the network was limitedlimited..
PROPOSED TRAINING ACTIVITIESPROPOSED TRAINING ACTIVITIES
Standardization and evaluation of clinical protocolsStandardization and evaluation of clinical protocols
Training on Diagnosis and clinical management of drug Training on Diagnosis and clinical management of drug resistant malariaresistant malaria
Training on safe practices and data quality assurance.Training on safe practices and data quality assurance.
Training on advanced HPLC analytical techniquesTraining on advanced HPLC analytical techniques
FUTURE PLANSFUTURE PLANS Development of a protocol for rapid diagnosis and detection of drug Development of a protocol for rapid diagnosis and detection of drug
resistant infections using other body fluids especially saliva. resistant infections using other body fluids especially saliva.
Acquisition and establishment of the technology for microsatelite and Acquisition and establishment of the technology for microsatelite and DNA arrays.DNA arrays.
Acquisition of HPLC technique for artemisinin derivatives, Acquisition of HPLC technique for artemisinin derivatives, mefloquine and halofanthrine.mefloquine and halofanthrine.
Summary of Summary of accomplishmentsaccomplishments Number of patients enrolled to date = 158Number of patients enrolled to date = 158
• CQ =CQ =• SP =SP =
Number completing 14 day follow up = 140Number completing 14 day follow up = 140• CQ =79CQ =79• SP =61SP =61
Number of samples analysed (molecular markers) =Number of samples analysed (molecular markers) =• dhfrdhfr• Pfcrt- 104Pfcrt- 104
Number of samples analysed (in vitro susceptibility) =Number of samples analysed (in vitro susceptibility) =• CQ = 80CQ = 80• SP = 20SP = 20
Number of samples analysed (drug levels in blood) =Number of samples analysed (drug levels in blood) =• CQ = CQ = • SP =SP =
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
WHO MIM/TDRWHO MIM/TDR All the patients who volunteered to participate in the study.All the patients who volunteered to participate in the study.
Walter Reed Army Institute of Research, Washington DC: Walter Reed Army Institute of Research, Washington DC: (Dr. Dennis Kyle).(Dr. Dennis Kyle).
Harvard School of Public Health: (Prof. Dyann Wirth)Harvard School of Public Health: (Prof. Dyann Wirth)
