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MIM/TDR DRUG RESISTANT NETWORK

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  • 1. MIM/TDR ANTIMALARIAL DRUG RESISTANTMIM/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 forInstitution: Malaria Research Laboratories, Postgraduate Institute for Medical Research and Training, College of Medicine, University ofMedical Research and Training, College of Medicine, University of Ibadan.Ibadan.
  • 2. OBJECTIVESOBJECTIVES SCIENTIFIC OBJECTIVESSCIENTIFIC OBJECTIVES:: • Definition of the characteristics ofDefinition of the characteristics of P. falciparumP. falciparum to chloroquine (CQ),to chloroquine (CQ), sulphadoxine-pyrimethamine (SP), amodiaquine (AQ), mefloquine (MQ),sulphadoxine-pyrimethamine (SP), amodiaquine (AQ), mefloquine (MQ), halofanthrine (HF) and artesunate/SP combination).halofanthrine (HF) and artesunate/SP combination). • Collation of clinical, parasitological (in vitro & molecular) andCollation of clinical, parasitological (in vitro & molecular) and pharmacokinetic data following treatment with Chloroquine orpharmacokinetic 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 malariaStrengthening 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.
  • 3. CAPACITY BUILDING OBJECTIVESCAPACITY BUILDING OBJECTIVES Establish capacity sustainable to implement the 5 main aspects of theEstablish 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 drugTrain 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
  • 4. PARTNERS AND COLLABORATINGPARTNERS AND COLLABORATING INSTITUTIONSINSTITUTIONS Walter Reed Army Institute of Research, USAWalter Reed Army Institute of Research, USA (Dr. Dennis Kyle)(Dr. Dennis Kyle) Harvard School of Public Health, USAHarvard School of Public Health, USA (Prof. Dyann Wirth)(Prof. Dyann Wirth) Thammasat University ThailandThammasat University Thailand (Dr. Kesara(Dr. Kesara Nabachang)Nabachang) Karolinska InstituteKarolinska Institute ((in discussionin discussion))
  • 5. BACKGROUND AND RATIONALE FORBACKGROUND AND RATIONALE FOR PROJECTPROJECT Chloroquine resistantChloroquine resistant P. falciparumP. falciparum infections representinfections represent a major public health problem in Nigeria.a major public health problem in Nigeria. In most areas where chloroquine resistance hasIn most areas where chloroquine resistance has emerged, treatment of malaria relies on the use ofemerged, treatment of malaria relies on the use of alternative antimalarial drugs including:alternative antimalarial drugs including: • Sulphadoxine-pyrimethamineSulphadoxine-pyrimethamine • AmodiaquineAmodiaquine • MefloquineMefloquine • Artemisinin derivativesArtemisinin derivatives • Halofanthrine.Halofanthrine.
  • 6. 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:
  • 7. 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 distributionThis 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..
  • 8. 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 ethicalStudy 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 symptomsPatients aged 6months to 5 years with clinical symptoms microscopically confirmedmicroscopically confirmed P. falciparumP. falciparum infection and wereinfection and were enrolled after informed consents from parent/guardian.enrolled after informed consents from parent/guardian.
  • 9. Clinical studies (cont’d)Clinical studies (cont’d) • Blood was obtained from each patient for hematology andBlood was obtained from each patient for hematology and biochemistry prior to treatment and selected time intervals.biochemistry prior to treatment and selected time intervals. • Patients were treated with standard doses of either chloroquine orPatients were treated with standard doses of either chloroquine or sulphadoxine-pyrimethamine.sulphadoxine-pyrimethamine. • Each patient was followed up for 14 days to monitor clinicalEach patient was followed up for 14 days to monitor clinical response to treatment.response to treatment.
  • 10. Parasite susceptibility and laboratory studiesParasite susceptibility and laboratory studies • Venous blood was obtained from each patient prior to treatmentVenous 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 drugsSusceptibility of patient isolates to standard antimalarial drugs was performed using:was performed using: – A modification of the standard WHO schizont inhibition assayA modification of the standard WHO schizont inhibition assay based on the incorporation of selected resistance reversing agentsbased on the incorporation of selected resistance reversing agents (verapamil).(verapamil). – The double Elisa assay (DELI) based on pLDH activityThe double Elisa assay (DELI) based on pLDH activity
  • 11. Molecular Assays:Molecular Assays: • Blood samples from each patient were blotted on filter paperBlood 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 untilFilter 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 theNested PCR AND RLFP analysis were used to evaluate the prevalence of mutations in the pfcrt and pfmdr1 genes ofprevalence of mutations in the pfcrt and pfmdr1 genes of isolates of P. falciparum obtained from patients beforeisolates of P. falciparum obtained from patients before treatment and during recrudescence of infection.treatment and during recrudescence of infection. • Genotypes ofGenotypes of Plasmodium falciparumPlasmodium falciparum population, werepopulation, were determined using polymorphic markers (MSP-1, MSP-2 anddetermined using polymorphic markers (MSP-1, MSP-2 and GLURP) prior to treatment and during recrudescence fromGLURP) prior to treatment and during recrudescence from filter paper samples.filter paper samples.
  • 12. Antimalarial drug blood levelsAntimalarial drug blood levels • 100ul blood samples were blotted on filter paper prior to100ul blood samples were blotted on filter paper prior to treatment and on days 1, 3, 7 and 14 after treatment and attreatment and on days 1, 3, 7 and 14 after treatment and at recrudescence, dried at room temperature and stored untilrecrudescence, dried at room temperature and stored until HPLC analysis.HPLC analysis. • 1 ml Venous blood samples were obtained from patients1 ml Venous blood samples were obtained from patients treated with sulphadoxine pyrimethamine and the redtreated with sulphadoxine pyrimethamine and the red blood cells were separated from plasma and both fractionsblood 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 drugThe primary objective of this protocol is to correlate drug concentration with the clinical response.concentration with the clinical response.
  • 13. RESULTSRESULTS Clinical Outcome:Clinical Outcome: 140 patients completed 14 day follow up in the140 patients completed 14 day follow up in the urban site. 71 patients received chloroquine while 61 patients receivedurban site. 71 patients received chloroquine while 61 patients received sulphadoxine-pyrimethamine.sulphadoxine-pyrimethamine. Infection in 48% of patients treated withInfection in 48% of patients treated with CQ responded to treatment while infection in 74% of patients treated withCQ 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 CQ SP
  • 14. 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% clinical outcome in vitro profile in vitro profile CQ Sensitive CQ resistant MQ sensitive MQ resistant
  • 15. 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 out come in vivo/ invit ro sensit ive in vivo/ in vit ro resist ant clinical resist ance
  • 16. Molecular Profiles:Molecular Profiles: Collation of sequence polymorphisms at codon 86 ofCollation of sequence polymorphisms at codon 86 of the pfmdr 1 gene in pretreatment samples and clinical response tothe pfmdr 1 gene in pretreatment samples and clinical response to chloroquine treatmentchloroquine treatment Clinical profileClinical profile Pfmdr 1Pfmdr 1 MutationMutation Y86Y86 Wild type alleleWild type allele N86N86 Mixed alleleMixed allele (Y86 + N86)(Y86 + N86) CQ SensitiveCQ Sensitive 39%39% 39%39% 21%21% CQ ResistantCQ Resistant 14%14% 57%57% 29%29%
  • 17. 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 Clinical resistance pret reat ment molecular profile 46% 43% 0% 10% 20% 30% 40% 50% clinical Clinical resistance Post- treatment molecular profile
  • 18. 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 vitro pfmdr 1 Y86
  • 19. CORRELATION OF TREATMENT FAILURE , BLOOD LEVELS ANDCORRELATION OF TREATMENT FAILURE , BLOOD LEVELS AND MOLECULAR PROFILESMOLECULAR PROFILES Patient IDPatient ID Day of failureDay of failure CQ blood levelCQ blood level Pfcrt allele (Do)Pfcrt allele (Do) Pfcrt allele atPfcrt allele at RecrudescenceRecrudescence 041041 D14D14 494.6494.6 MIXEDMIXED T76T76 073073 D10D10 679.2679.2 T76T76 T76T76 O68O68 D14D14 880.0880.0 T76T76 T76T76 045045 D4D4 917.6917.6 MIXEDMIXED MIXEDMIXED 013013 D14D14 1032.81032.8 K76K76 K76K76 138138 D14D14 1421.31421.3 T76T76 T76T76 116116 D14D14 1665.91665.9 MIXEDMIXED MIXEDMIXED 087087 D18D18 1670.11670.1 T76T76 T76T76 024024 D15D15 1677.91677.9 T76T76 MIXEDMIXED 010010 D14D14 1825.71825.7 MIXEDMIXED T76T76 107107 D14D14 1873.41873.4 T76T76 T76T76 020020 D21D21 2243.22243.2 T76T76 K76K76
  • 20. Blood level of chloroquine in patients whose infectionBlood level of chloroquine in patients whose infection responded to chloroquine treatmentresponded to chloroquine treatment PATIENT IDPATIENT ID CHLOROQUINE BLOOD LEVEL (ng/ml)CHLOROQUINE BLOOD LEVEL (ng/ml) 127127 181.0181.0 098098 236.7236.7 007007 328.9328.9 022022 477.7477.7 017017 548.5548.5 147147 594.5594.5 016016 647.7647.7 047047 814.7814.7 123123 1111.91111.9 043043 1151.01151.0
  • 21. Patients with treatment failure:Patients with treatment failure: The concentration of CQ ranged from 494.6ng/ml to 2243.2ng/ml (mean CQThe 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 inThe 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 mutant58% 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
  • 22. MILESTONES & TIMELINES for 2nd yearMILESTONES & TIMELINES for 2nd year Completion of microscopic evaluation and data analysis of in vitroCompletion of microscopic evaluation and data analysis of in vitro studies conducted in 2002 and initiation of analysis of 2003 studiesstudies 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 theCompletion of molecular assays of all samples obtained from the studies conducted in 2002 and initiation of analysis of 2003 studiesstudies 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 initiationPharmacokinetic analysis of samples collected in 2002 and initiation of analysis of 2003 studiesof analysis of 2003 studies (January 2003 to November 2003)(January 2003 to November 2003) Training on Analytical techniques for each network siteTraining on Analytical techniques for each network site (May to(May to November 2003)November 2003) Training on Standardization and evaluation of clinical protocolsTraining on Standardization and evaluation of clinical protocols (April 2003)(April 2003)
  • 23. MILESTONES & TIMELINES (CONT’D)MILESTONES & TIMELINES (CONT’D) Group Training on Diagnosis and clinical managementGroup Training on Diagnosis and clinical management of drug resistant malariaof drug resistant malaria (April 2003)(April 2003) Patient recruitment and Clinical studies at The villagePatient 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 dataCollation and analysis of clinical data (September(September 2003 to November 2003)2003 to November 2003) Progress Report writingProgress Report writing (November 2003)(November 2003)
  • 24. 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 Republic Yomi 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
  • 25. CAPACITY BUILT (Year 1)CAPACITY BUILT (Year 1) Name of trainee LevelName of trainee Level SpecialtySpecialty DurationDuration Year of enrolmentYear of enrolment Mr. AdegokeMr. Adegoke ++ MSc Parasite biologyMSc Parasite biology 2years.2years. 20022002 Mr. Tanimowo MSc Analytical tech.Mr. Tanimowo MSc Analytical tech. 2years2years 20022002 Mr. RajiMr. Raji *MSc*MSc Molecular biologyMolecular biology 2 years2 years 20022002 Miss. Oduola *PhDMiss. Oduola *PhD Parasite biology &Parasite biology & 4 years4 years 20012001 Onikepe Olunloyo *PhDOnikepe Olunloyo *PhD Mol. BiolMol. Biol 4 Years4 Years 19991999 Abiola Olukosi *PhDAbiola Olukosi *PhD Mol. BiolMol. Biol 4 years4 years 19991999 Abayomi SijuadeAbayomi Sijuade *PhD Parasite biology &*PhD Parasite biology & 4 years4 years 20032003 Analytical tech.Analytical tech. • *Institution of training: College of Medicine, University of Ibadan*Institution of training: College of Medicine, University of Ibadan
  • 26. 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
  • 27. ENABLING FACTORS AND CHALLENGESENABLING FACTORS AND CHALLENGES Facilitating factors:Facilitating factors: The involvement of a network management team and other technicalThe 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 solvingThe 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 anThe 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 fromFinancial 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.
  • 28. ENABLING FACTORS AND CHALLENGES IIENABLING FACTORS AND CHALLENGES II Managment problems:Managment problems: The lack of fundsThe lack of funds was a limiting factor in projectwas a limiting factor in project execution. This was important as large part of theexecution. 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 forThe prolonged waiting periods for replacement of faulty equipment parts limited somereplacement of faulty equipment parts limited some aspects of the research activities especially the analysisaspects of the research activities especially the analysis of drugs in the patients samples.of drugs in the patients samples. Lack of functional uninterruptible internet connectivityLack of functional uninterruptible internet connectivity also confounded some aspects of the research effort asalso confounded some aspects of the research effort as contact with other investigators on the network wascontact with other investigators on the network was limitedlimited..
  • 29. PROPOSED TRAINING ACTIVITIESPROPOSED TRAINING ACTIVITIES Standardization and evaluation of clinical protocolsStandardization and evaluation of clinical protocols Training on Diagnosis and clinical management of drugTraining 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
  • 30. FUTURE PLANSFUTURE PLANS Development of a protocol for rapid diagnosis and detection of drugDevelopment 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 andAcquisition 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.
  • 31. Summary of accomplishmentsSummary of accomplishments 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 =
  • 32. 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)

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