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

The genetic change in P. falciparum populations of rural Tanzania resulting from national policy on firstline malaria treatment and pilot Sulfadoxine/pyrimethamine and Artesunate combination

Malaria Journal 2010, 9(Suppl 2):P20Theory predicts that we can protect the efficacy of future antimalarials by changing treatment practice or drug formulation, but the potential success of such interventions rests upon their impact on drug pressure in the field. So far, gathering field data on the relationship between policy, drug pressure, recombination and the evolution of resistance has been entirely challenging. To test these predictions, dhfr and dhps frequency changes were measured in two rural districts of Rufiji and Kilombero/Ulanga during 2000-2006, and the frequencies of the two genes compared prior, during and after antimalarial policy change from first line CQ to first line SP in 2001. Furthermore, while SP first line was maintained in Kilombero/Ulanga, pilot combination therapy of SP+Artesunate (ART) was introduced in Rufiji in 2002 to replace SP and dhfr and dhps frequency changes compared between the two districts. Size polymorphisms at three sets of microsatellite loci linked to dhfr and three other sets of unlinked microsatellite loci were analysed. Genetic analysis of SP resistance genes was carried out on 9,662 Plasmodium falciparum infections identified in a series of annual cross sectional surveys conducted in the two districts between 2000-2006. The frequency of dhfr and dhps resistance alleles did not change significantly while SP was the recommended second-line treatment, but highly significant changes occurred during the subsequent year after the switch to first line SP. The frequency of the triple mutant dhfr allele increased by 37% -63% and that of double mutant dhps allele increased 200%-300%. A strong association between these unlinked alleles also emerged; confirming that they are co-selected by SP. Distribution of major lineages indicates that there is extensive genetic exchange among the geographic regions. Combination therapy had visible effect on the frequencies of dhfr and dhps resistance alleles. The findings of this study provide insight on the interplay between policy, drug pressure, recombination and the evolution of resistance

Quantification of markers of antimalarial drug resistance from an area of high malaria transmission: Comparing frequency with prevalence

Molecular monitoring of markers of antimalarial drug resistance offers an affordable alternative to the in vivo method for the detection of resistance, and has the potential to guide public health policy in a timely manner. However, the optimal way of analyzing and reporting these data, particularly those emanating from areas of moderate to high malaria transmission, has never been fully explored or agreed upon, given the potential of being confounded by coinfections. By using large number of real field samples, we quantified the difference between prevalence and frequency when reporting field data on antimalarial drug resistance obtained by direct counting of haplotypes. Polymerase chain reaction (PCR) and sequence specific oligonucleotide probing was used to generate point mutations which were used to construct haplotypes. Results indicate that frequency underestimates haplotypes present at low levels while also amplifying haplotypes present at high levels; prevalence on the other hand behaved in a vice versa manner. Both prevalence and frequency are therefore essential, as each may have relevance in different contexts in high malaria transmission settings. Frequency is essential to gauge the impact of intervention on antimalarial drug resistance while prevalence may be more relevant when the aim is to determine parasite clearance. Key words: Molecular markers, polymerase chain reaction (PCR) - sequence specific oligonucleotide probing (SSOP), prevalence, frequency

Media, Health Workers, and Policy Makers' Relationship and Their Impact on Antimalarial Policy Adoption: A Population Genetics Perspective

Drug resistance negatively impacts malaria treatments, making treatment policy revision unavoidable. So far, studies relating sociopolitical and technical issues on policy change with malaria parasite genetic change are lacking. We have quantified the effect of malaria treatment policy on drug pressure and the influence of the media, policy makers, and health worker relationship on parasite population genetic change in Kilombro/Ulanga district. Cross-sectional surveys of asymptomatic infections conducted before, during and after the switch from chloroquine to sulphadoxine/pyrimethamine were used for genetic analysis of SP resistance genes in 4,513 asymptomatic infections identified, and their frequency change was compared with retrospective study of the documented process of policy change. Highly significant changes of dhfr and dhps resistance alleles occurred within one year of switch to SP first line, followed by a decline of their rate of selection caused by reduction of SP usage, as a result of negative media reports on SP usage and lack of adequate preparations

The evolution of pyrimethamine resistant dhfr in Plasmodium falciparum of south-eastern Tanzania: comparing selection under SP alone vs SP+artesunate combination

BACKGROUND\ud \ud Sulphadoxine-pyrimethamine (SP) resistance is now widespread throughout east and southern Africa and artemisinin compounds in combination with synthetic drugs (ACT) are recommended as replacement treatments by the World Health Organization (WHO). As well as high cure rates, ACT has been shown to slow the development of resistance to the partner drug in areas of low to moderate transmission. This study looked for evidence of protection of the partner drug in a high transmission African context. The evaluation was part of large combination therapy pilot implementation programme in Tanzania, the Interdisciplinary Monitoring Programme for Antimalarial Combination Therapy (IMPACT-TZ) METHODS: The growth of resistant dhfr in a parasite population where SP Monotherapy was the first-line treatment was measured for four years (2002-2006), and compared with the development of resistant dhfr in a neighbouring population where SP + artesunate (SP+AS) was used as the first-line treatment during the same interval. The effect of the differing treatment regimes on the emergence of resistance was addressed in three ways. First, by looking at the rate of increase in frequency of pre-existing mutant dhfr alleles under monotherapy and combination therapy. Second, by examining whether de-novo mutant alleles emerged under either treatment. Finally, by measuring diversity at three dhfr flanking microsatellite loci upstream of the dhfr gene.\ud \ud RESULTS\ud \ud The reduction in SP selection pressure resulting from the adoption of ACT slowed the rate of increase in the frequency of the triple mutant resistant dhfr allele. Comparing between the two populations, the higher levels of genetic diversity in sequence flanking the dhfr triple mutant allele in the population where the ACT regimen had been used indicates the reduction in SP selection pressure arising from combination therapy.\ud \ud CONCLUSION\ud \ud The study demonstrated that, alleles containing two mutations at the dhfr have arisen at least four times independently while those containing triple mutant dhfr arose only once, and were found carrying a single unique Asian-type flanking sequence, which apparently drives the spread of pyrimethamine resistance associated dhfr alleles in east Africa. SP+AS is not recommended for use in areas where SP cure rates are less than 80% but this study reports an observed principle of combination protection from an area where pyrimethamine resistance was already high

Multiple Origins and Regional Dispersal of Resistant dhps in African Plasmodium falciparum Malaria

Cally Roper and colleagues analyze the distribution of sulfadoxine resistance mutations and flanking microsatellite loci to trace the emergence and dispersal of drug-resistant Plasmodium falciparum malaria in Africa

Drug resistance to sulphadoxine-pyrimethamine in Plasmodium falciparum malaria in Mlimba, Tanzania

BACKGROUND: Sulphadoxine-pyrimethamine (SP) has been and is currently used for treatment of uncomplicated Plasmodium falciparum malaria in many African countries. Nevertheless, the response of parasites to SP treatment has shown significant variation between individuals. METHODS: The genes for dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) were used as markers, to investigate parasite resistance to SP in 141 children aged less than 5 years. Parasite DNA was extracted by Chelex method from blood samples collected and preserved on filter papers. Subsequently, polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP) were applied to detect the SP resistance-associated point mutations on dhfr and dhps. Commonly reported point mutations at codons 51, 59, 108 and 164 in the dhfr and codons 437, 540 and 581 in the dhps domains were examined. RESULTS: Children infected with parasites harbouring a range of single to quintuple dhfr/dhps mutations were erratically cured with SP. However, the quintuple dhfr/dhps mutant genotypes were mostly associated with treatment failures. High proportion of SP resistance-associated point mutations was detected in this study but the adequate clinical response (89.4%) observed clinically at day 14 of follow up reflects the role of semi-immunity protection and parasite clearance in the population. CONCLUSION: In monitoring drug resistance to SP, concurrent studies on possible confounding factors pertaining to development of resistance in falciparum malaria should be considered. The SP resistance potential detected in this study, cautions on its useful therapeutic life as an interim first-line drug against malaria in Tanzania and other malaria-endemic countries

Drug coverage in treatment of malaria and the consequences for resistance evolution - evidence from the use of sulphadoxine/pyrimethamine

BACKGROUND\ud \ud It is argued that, the efficacy of anti-malarials could be prolonged through policy-mediated reductions in drug pressure, but gathering evidence of the relationship between policy, treatment practice, drug pressure and the evolution of resistance in the field is challenging. Mathematical models indicate that drug coverage is the primary determinant of drug pressure and the driving force behind the evolution of drug resistance. These models show that where the basis of resistance is multigenic, the effects of selection can be moderated by high recombination rates, which disrupt the associations between co-selected resistance genes.\ud \ud METHODS\ud \ud To test these predictions, dhfr and dhps frequency changes were measured during 2000-2001 while SP was the second-line treatment and contrasted these with changes during 2001-2002 when SP was used for first-line therapy. Annual cross sectional community surveys carried out before, during and after the policy switch in 2001 were used to collect samples. Genetic analysis of SP resistance genes was carried out on 4,950 Plasmodium falciparum infections and the selection pressure under the two policies compared.\ud \ud RESULTS\ud \ud The influence of policy on the parasite reservoir was profound. The frequency of dhfr and dhps resistance alleles did not change significantly while SP was the recommended second-line treatment, but highly significant changes occurred during the subsequent year after the switch to first line SP. The frequency of the triple mutant dhfr (N51I,C59R,S108N) allele (conferring pyrimethamine resistance) increased by 37% - 63% and the frequency of the double A437G, K540E mutant dhps allele (conferring sulphadoxine resistance) increased 200%-300%. A strong association between these unlinked alleles also emerged, confirming that they are co-selected by SP.\ud \ud CONCLUSION\ud \ud The national policy change brought about a shift in treatment practice and the resulting increase in coverage had a substantial impact on drug pressure. The selection applied by first-line use is strong enough to overcome recombination pressure and create significant linkage disequilibrium between the unlinked genetic determinants of pyrimethamine and sulphadoxine resistance, showing that recombination is no barrier to the emergence of resistance to combination treatments when they are used as the first-line malaria therapy

Prevalence and constraints of typhoid fever and its control in an endemic area of Singida region in Tanzania: Lessons for effective control of the disease

Journal of Public Health and Epidemiology Vol. 2(5), pp. 93-99, August 2010Typhoid fever (TF) is an important communicable disease that is endemic to Tanzania, quite often complicated with malaria co-infection leading to diagnostic complications and significant mortality. Despite considerable control efforts, the disease continues to persist in Singida urban leading to significant morbidity and mortality. This paper retrospectively examines morbidity and mortality trend of typhoid infection for the past five years in Singida urban. By using questionnaire, Focus Group Discussion (FGD) and direct observational methods, the authors report interview results of 120 respondents from the study community, regarding their awareness of TF and its control methods. Results showed that, the TF prevalence records revealed a fluctuating trend with annual incidence rate of 580 – 1,400/100,000 persons, and an overall increase from 771 – 942 cases/100,000 persons (p _ 0.0001) between 2003 and 2007. While 88% of the respondents were aware of TF disease, 53% were unaware of its control methods. The study also revealed an acute shortage of diagnostic laboratory services which indicated that, 75% of health facilities had no such services. In adequate knowledge about personal hygiene, scarcity or lack of access to safe water, improper drainage systems and problems of unsanitary toilets in Singida urban were some of the obstacles to effective TF control. Effective TF control measures in the study district, as in other areas in the tropics, requires integration of intensive health education as a public health tool, provision and access to safe water supply and adequate strengthening of health systems