"Plasmodium falciparum" Drug Resistance, Molecular Genotyping and Generation of a Malaria Resistance Genogram by DNA Microarray-Based Technology

Prior to the 2001 malarial treatment policy change in Tanzania, studies were conducted to assess the efficacy of sulfadoxine-pyrimethamine (SP) and usefulness of molecular markers in monitoring SP resistance. In these studies the 1996 WHO protocol (with 14 days follow-up) was used to assess treatment responses. The findings show that SP failure rates were 6.8 – 13.5% and P. falciparum triple-Pfdhfr mutant genotype (18.6 – 21.8 %) were already prevalent prior to the change. Mkuzi site, which due to high CQ failure rate, had been using SP against pediatric malaria since 1984, had exceptionally high failure rate (23.6%) and prevalence of triple-Pfdhfr mutant genotype (80%). Therefore, the study suggested that the drug may have a short useful therapeutic life (UTL) in Tanzania. Hence SP was adopted as an interim first line antimalarial drug in 2001 while combination therapies were being evaluated for long-term use. The molecular findings also pointed to the potential of the triple-pfdhfr mutant genotype as an early warning tool for increasing SP resistance. These data formed the baseline SP efficacy and molecular markers profile in Tanzania prior to the policy change. SP efficacy monitoring studies conducted using the WHO 2002 protocol (with 28 days follow-up) after widespread use of SP showed high (~40%) SP failure rates in Tanzania. Therefore, these findings provided evidence for withdrawing SP use in Tanzania and highly justified the recommendation of Ministry of Health of switching the first line treatment to artemether-lumefantrine (AL). Concurrent with efficacy studies, community surveys were conducted in the health facilities’ catchment areas to assess the profile of all molecular markers of P. falciparum resistance to withdrawn and in-use antimalarial drugs as well as those that have never been officially deployed for use. Results show that molecular markers of SP resistance are more prevalent compared to those recorded prior to adoption of SP, with triple-Pfdhfr mutant genotype ranging from 54 – 74%. The triple- Pfdhfr mutant genotype showed some little evidence of depiction of SP failure rate observed at health facilities. This marker may be applicable as a tool in community-based surveillance of dynamics of SP resistance. However, its usefulness must be further explored by assessing its dynamics relative to SP failure rates in many sites, preferably with different failure rates while taking into account linkage disequilibria of the observed mutations. The frequencies of the main mediators of CQ resistance markers (Pfcrt 76 and Pfmdr1 86 Asn) have decreased following suspension of CQ use in Tanzania, but seems to be influenced by uncontrolled use of QN and AQ for uncomplicated malaria. The lack of the ATPase6 mutation suggest that resistance to artemisinin has not been selected in Tanzania. This observation further supports the decision to adopt AL. The capacity to detect many SNPs in many resistance conferring genes makes DNA micorarray technology a potential tool for monitoring dynamics of P falciparum resistance to both withdrawn, “in-use” and undeployed drugs. However, in order to better appreciate parasite genotypic dynamics following alteration of drug pressure, the interval between assessments should be relatively longer than the one used in this study. In the framework of WHO/TDR combination therapy (ACT) trials of uncomplicated P. falciparum malaria in Sub-Sahara African children, we assessed genotyping performance and use-effectiveness. Parasitological failures were adjusted by stepwise genotyping the P. falciparum glutamine rich protein (glurp), merozoite surface protein 1 (msp1) and 2 (msp2) in Day 0 and post-Day14 recurrent parasitaemias. Recurrences on or before Day 14 were assumed to be recrudescent and were not genotyped. Molecular genotyping refined parasitological outcomes, with differences between crude and adjusted outcomes in most sites >10%. The overall and laboratory performances, (69%) and (78%), respectively, of the stepwise genotyping system were better. However, diligence is needed in sample collection and analysis in order to reduce loss of genotyping data and hence failure to resolve recurrences. Additional genotyping of pre-Day 14 recurrences in Uganda site identified many more new infections and further reduced the PCR-adjusted parasitological failure rate by 8%. Therefore, the study recommends that all recurrent infections in malaria treatment trials/studies in high transmission areas should be genotyped. The stepwise genotyping approach, coupled with more advanced DNA extraction methods needs to be validated and considered for adoption as a standard integral part in malaria drug efficacy studies

Exploration of in Vivo Efficacy of Artemether-Lumefantrine Against Uncomplicated Plasmodium Falciparum Malaria in Under Fives in Tabora Region, Tanzania.

Tanzania adopted artemether-lumefantrine (AL) as first-line drug for uncomplicated malaria in 2006. Recently, there was an anecdotal report on high malaria recurrence rate following AL treatment in in the (urban and peri-urban), western part of Tanzania. The current report is an exploratory study to carefully and systematically assess AL efficacy in the area. Between June and August 2011, a total of 1,126 patients were screened for malaria, 33 had malaria, of which 20 patients met inclusion criteria and were enrolled and treated with standard dose of AL as recommended in the WHO protocol. Treated patients were followed up for 28 days to assess treatment responses. Before treatment (Day 0) and post-treatment (Day 7) plasma lumefantrine levels were determined to assess prior AL use and ascertain parasites exposure to adequate plasma leveles of lumefantrine, respectively. The cure rate was 100%. All Day 0 plasma lumefantrine were below HPLC detectable level. The median Day 7 lumefantrine concentration was 404, (range, 189-894 ng/ml). Six out of 20 patients (30%) were gametocytaemic and all cleared gametocytes by Day 14. One patient showed an increase in gametocytes from four on Day 0 to 68, per 500 WBC on Day 2. Artemether lumefantrine is highly efficacious against uncomplicated Plasmodium falciparum malaria. The elevation of gametocytaemia despite AL treatment needs to be evaluated in a larger study

Influencing innovation structures and processes in agro-industries dominated by subsistence producers: an analysis of the rural poultry industry in Tanzania

This thesis examines innovation structures and processes in rural poultry industry in Tanzania. In 2005, FAO categorised the rural poultry production system in Tanzania under the lowest sector IV with very minimal biosecurity measures and with no commercial orientation. By 2012, a DFID-funded Research into Use (RIU) programme transformed the industry to Sector III which represents a significant commercial orientation and relatively higher bio-security measures. This thesis explains how RIU achieved that. This analysis is presented from three perspectives. First, the path dependence framework is used to present the observed dominance of the traditional poultry production system as a ‘lock-in’. The study makes it clear that before RIU, mental frames, resource allocations and how dominant powers behaved reinforced low innovation tendencies. Second, using the agricultural innovation system (AIS) framework and the concepts of ‘organisational thinness’ and ‘fragmentation’ (also from path dependency theory), it explains that by making rural producers feel self-sufficient in inputs and knowledge, practices in the traditional system disconnect producers from engaging with other actors. Third, the concepts of ‘innovation broker’ and of ‘exogenous shock’ are used to present RIU as an external force or facilitator which instigated a transformation process. RIU facilitated a large number of rural producers to produce for the market, and which was sufficient enough to create a significant demand for inputs and services. This demand triggered new investment and re-organisation in the supply chains. Then, RIU supported actors to solve capacity problems that emerged from the shock. RIU is therefore presented as a flexible ‘innovation broker’ who played different roles and allocated resources based on circumstances on the ground. The thesis makes several contributions. It presents a case of how a public action can promote innovation in industries dominated by subsistence producers by playing the role of an innovation broker to support a significant number of producers to change routines and interact with other actors. It also shows that rural growth can be achieved through linking rural enterprises with those in the urban instead of supporting rural actors in isolation. It basically makes it clear that African agriculture needs re-organization, so that technological changes can follow as a consequence

Efficacy of sulfadoxine-pyrimethamine in Tanzania after two years as first-line drug for uncomplicated malaria: assessment protocol and implication for treatment policy strategies.

BACKGROUND\ud \ud Systematic surveillance for resistant malaria shows high level of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) across eastern and southern parts of Africa. This study assessed in vivo SP efficacy after two years of use as an interim first-line drug in Tanzania, and determined the rates of treatment failures obtained after 14 and 28 days of follow-up.\ud \ud METHODS\ud \ud The study was conducted in the Ipinda, Mlimba and Mkuranga health facilities in Tanzania. Children aged 6-59 months presenting with raised temperature associated exclusively with P. falciparum (1,000-100,000 parasites per microl) were treated with standard dose of SP. Treatment responses were classified according to the World Health Organization (WHO) definition as Adequate Clinical and Parasitological Response (ACPR), Early Treatment Failure (ETF), Late Clinical Failure (LCF) and Late Parasitological Failure (LPF) on day 14 and day 28.\ud \ud RESULTS\ud \ud Overall 196 (85.2%) of 230 patients had ACPR on day 14 but only 116 (50.9%) on day 28 (57.7% after excluding new infections by parasite genotyping). Altogether 21 (9.1%) and 13 (5.7%) of the 230 patients assessed up to day 14 and 39 (17.1%) and 55 (24.1%) of the 228 followed up to day 28 had clinical and parasitological failure, respectively.\ud \ud CONCLUSION\ud \ud These findings indicate that SP has low therapeutic value in Tanzania. The recommendation of changing first line treatment to artemether + lumefantrine combination therapy from early next year is, therefore, highly justified. These findings further stress that, for long half-life drugs such as SP, establishment of cut-off points for policy change in high transmission areas should consider both clinical and parasitological responses beyond day 14

Therapeutic Efficacy of Sulfadoxine-Pyrimethamine and Prevalence of Resistance Markers in Tanzania Prior to Revision of Malaria Treatment Policy: Plasmodium Falciparum Dihydrofolate Reductase and Dihydropteroate Synthase Mutations in Monitoring in Vivo Resistance.

Prior to the 2001 malarial treatment policy change in Tanzania, we conducted trials to assess the efficacy of sulfadoxine-pyrimethamine (SP) and the usefulness of molecular markers in monitoring resistance. A total of 383 uncomplicated Plasmodium falciparum malaria patients (between 6 and 59 months old) were treated with SP and their responses were assessed. Mutations in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes in admission day blood samples were analyzed. Results indicated that 85.6% of the patients showed an adequate clinical response, 9.7% an early treatment failure, and 4.7% a late treatment failure. The quintuple mutant genotype (pfdhfr 51 Ile, 59 Arg, and 108 Asn and pfdhps 437 Gly and 540 Glu) showed an association with treatment outcome (odds ratio = 2.1; 95% confidence interval = 0.94-4.48, P = 0.045). The prevalence of the triple pfdhfr mutant genotype (51 Ile, 59 Arg, and 108 Asn) at a site of high SP resistance (23.6%) was four times higher compared with that observed at sites of moderate SP resistance (6.8-14.4%) (P = 0.000001). The genotype failure index calculated by using this marker was invariable (1.96-2.1) at sites with moderate SP resistance, but varied (3.4) at a site of high SP resistance. In conclusion, our clinical and molecular findings suggest that SP may have a short useful therapeutic life in Tanzania; thus, its adoption as an interim first-line antimalarial drug. The findings also point to the potential of the triple pfdhfr mutant genotype as an early warning tool for increasing SP resistance. These data form the baseline SP efficacy and molecular markers profile in Tanzania prior to the policy change

Efficacy and safety of artemisinin-based antimalarial in the treatment of uncomplicated malaria in children in southern Tanzania

BACKGROUND\ud \ud Tanzania switched the antimalarial first line to sulphadoxine-pyrimethamine (SP) in 2001 from ineffective chloroquine (CQ). By 2003 higher levels of SP resistance were recorded, prompting an urgent need for replacing the first line drug with ACT, as currently recommended by the World Health Organization. Despite this recommendation country-specific evidence-based data to support efficacy and safety profile of ACT is still limited. A study on the efficacy and safety of artesunate plus amodiaquine (AS+AQ) and artemether plus lumefantrine (AL)(Coartem) was carried out in 2004 with the view of supporting the National Malaria Control Programme in the review of the policy in mainland Tanzania.\ud \ud METHODS\ud \ud An in vivo efficacy study was conducted at Ipinda and Mlimba health facilities between May and November 2004. The study recruited children aged 6-59 months presenting with symptoms of uncomplicated malaria, history of fever or an axillary temperature > or =37.5 degrees C; mono infection with Pasmodium falciparum (2,000-200,000 parasites/microl). Patients were randomized to received either SP or amodiaquine monotherapy or treated with standard doses of AS+AQ in Mlimba and Coartem in Kyela and followed-up for 28 days to assess treatment responses. This study reports results of the combination therapies.\ud \ud RESULTS\ud \ud A total of 157 children (76 in Mlimba and 99 in Kyela) who were enrolled in to the study and treated with either AL or AS+AQ were successfully followed-up. Both combinations were tolerated and effected rapid fever and parasite clearance. The crude ACPRs were 80 (87%) and 41 (63%) for AL and AS+AQ respectively. However, after PCR adjustments the corresponding figures raised to 100% (n = 86) and 93.8% (n = 45) in AL and AS+AQ groups, respectively. The mean haemoglobin improved moderately from day 0 to day 28 by 1 g/dl in AL and 0.4 g/dl in AS+AQ treatment group and was statistically significant (p < 0.001 both).\ud \ud CONCLUSION\ud \ud These findings provide substantial evidence that AL is highly efficacious in areas of high resistance of SP and supported the country's decision to switch from SP monotherapy to AL

The role of pfmdr1 in Plasmodium falciparum tolerance to artemether-lumefantrine in Africa

Objective Artemether-lumefantrine (AL), presently the most favoured combination therapy against uncomplicated Plasmodium falciparum malaria in Africa, has recently shown to select for the pfmdr1 86N allele. The objective of this study was to search for the selection of other mutations potentially involved in artemether-lumefantrine tolerance and/or resistance, i.e. pfmdr1 gene amplification, pfmdr1 Y184F, S1034C, N1042D, D1246Y, pfcrt S163R and PfATP6 S769N. Methods The above mentioned SNPs were analysed by PCR-restriction fragment length polymorphism and pfmdr1 gene amplification by real-time PCR based protocols in parasites from 200 children treated with AL for uncomplicated P. falciparum malaria in Zanzibar. Results A statistically significant selection of pfmdr1 184F mostly in combination with 86N was seen in reinfections after treatment. No pfmdr1 gene amplification was found. Conclusion The results suggest that different pfmdr1 alleles are involved in the development of tolerance/resistance to lumefantrine.info:eu-repo/semantics/publishedVersio

Molecular monitoring of Plasmodium falciparum resistance to artemisinin in Tanzania

Artemisinin-based combination therapies (ACTs) are recommended for use against uncomplicated malaria in areas of multi-drug resistant malaria, such as sub-Saharan Africa. However, their long-term usefulness in these high transmission areas remains unclear. It has been suggested that documentation of the S769N PfATPase6 mutations may indicate an emergence of artemisinin resistance of Plasmodium falciparum in the field. The present study assessed PfATPase6 mutations (S769N and A623E) in 615 asymptomatic P. falciparum infections in Tanzania but no mutant genotype was detected. This observation suggests that resistance to artemisinin has not yet been selected in Tanzania, supporting the Ministry of Health's decision to adopt artemether+lumefantrine as first-line malaria treatment. The findings recommend further studies to assess PfATPase6 mutations in sentinel sites and verify their usefulness in monitoring emergency of ACT resistance

Plasmodium falciparum msp1, msp2 and glurp allele frequency and diversity in sub-Saharan Africa

The efficacy of anti-malarial drugs is assessed over a period of 28-63 days (depending on the drugs' residence time) following initiation of treatment in order to capture late failures. However, prolonged follow-up increases the likelihood of new infections depending on transmission intensity. Therefore, molecular genotyping of highly polymorphic regions of Plasmodium falciparum msp1, msp2 and glurp loci is usually carried out to distinguish recrudescence (true failures) from new infections. This tool has now been adopted as an integral part of anti-malarial efficacy studies and clinical trials. However, there are concerns over its utility and reliability because conclusions drawn from molecular typing depend on the genetic profile of the respective parasite populations, but this profile is not systematically documented in most endemic areas. This study presents the genetic diversity of P. falciparum msp1, msp2 and glurp markers in selected sub-Saharan Africa countries with varying levels of endemicity namely Malawi, Tanzania, Uganda, Burkina Faso and São Tomé.A total 780 baseline (Day 0) blood samples from children less than seven years, recruited in a randomized controlled clinical trials done between 1996 and 2000 were genotyped. DNA was extracted; allelic frequency and diversity were investigated by PCR followed by capillary electrophoresis for msp2 and fragment sizing by a digitalized gel imager for msp1 and glurp. Plasmodium falciparum msp1, msp2 and glurp markers were highly polymorphic with low allele frequencies. A total of 17 msp1 genotypes [eight MAD20-, one RO33- and eight K1-types]; 116 msp2 genotypes [83 3D7 and 33 FC27- types] and 14 glurp genotypes were recorded. All five sites recorded very high expected heterozygosity (HE) values (0.68 - 0.99). HE was highest in msp2 locus (HE=0.99), and lowest for msp1 (HE=0.68) (P<0.0001). The genetic diversity and allelic frequency recorded were independent of transmission intensity (P=0.84, P=0.25 respectively. A few genotypes had particularly high frequencies; however the most abundant showed only a 4% probability that a new infection would share the same genotype as the baseline infection. This is unlikely to confound the distinction of recrudescence from new infection, particularly if more than one marker is used for genotyping. Hence, this study supports the use of msp1, msp2 and glurp in malaria clinical trials in sub-Saharan Africa to discriminate new from recrudescent infections

Different methodological approaches to the assessment of in vivo efficacy of three artemisinin-based combination antimalarial treatments for the treatment of uncomplicated falciparum malaria in African children.

BACKGROUND: Use of different methods for assessing the efficacy of artemisinin-based combination antimalarial treatments (ACTs) will result in different estimates being reported, with implications for changes in treatment policy. METHODS: Data from different in vivo studies of ACT treatment of uncomplicated falciparum malaria were combined in a single database. Efficacy at day 28 corrected by PCR genotyping was estimated using four methods. In the first two methods, failure rates were calculated as proportions with either (1a) reinfections excluded from the analysis (standard WHO per-protocol analysis) or (1b) reinfections considered as treatment successes. In the second two methods, failure rates were estimated using the Kaplan-Meier product limit formula using either (2a) WHO (2001) definitions of failure, or (2b) failure defined using parasitological criteria only. RESULTS: Data analysed represented 2926 patients from 17 studies in nine African countries. Three ACTs were studied: artesunate-amodiaquine (AS+AQ, N = 1702), artesunate-sulphadoxine-pyrimethamine (AS+SP, N = 706) and artemether-lumefantrine (AL, N = 518).Using method (1a), the day 28 failure rates ranged from 0% to 39.3% for AS+AQ treatment, from 1.0% to 33.3% for AS+SP treatment and from 0% to 3.3% for AL treatment. The median [range] difference in point estimates between method 1a (reference) and the others were: (i) method 1b = 1.3% [0 to 24.8], (ii) method 2a = 1.1% [0 to 21.5], and (iii) method 2b = 0% [-38 to 19.3].The standard per-protocol method (1a) tended to overestimate the risk of failure when compared to alternative methods using the same endpoint definitions (methods 1b and 2a). It either overestimated or underestimated the risk when endpoints based on parasitological rather than clinical criteria were applied. The standard method was also associated with a 34% reduction in the number of patients evaluated compared to the number of patients enrolled. Only 2% of the sample size was lost when failures were classified on the first day of parasite recurrence and survival analytical methods were used. CONCLUSION: The primary purpose of an in vivo study should be to provide a precise estimate of the risk of antimalarial treatment failure due to drug resistance. Use of survival analysis is the most appropriate way to estimate failure rates with parasitological recurrence classified as treatment failure on the day it occurs