Plasmodium falciparum dihydrofolate reductase Val-16 and Thr-108 mutation associated with in vivo resistance to antifolate drug: a case study

Due to increasing trend in chloroquine resistance, the antifolate (sulpha-pyrimethamine combination) drugs are gaining more importance in the treatment of uncomplicated falciparum malaria. The efficacy of sulpha-pyrimethamine combinations in the treatment is compromised by the development of resistance in parasite. The occurrence of mutations at active sites in Plasmodium falciparum gene sequences coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) confer resistance to pyrimethamine and sulphadoxine. This study presents the characterization of a P. falciparum sample from a patient who did not respond to standard doses of a pyrimethamine/sulpha regimen. Although parasitaemia fell rapidly, the infection had not resolved six days later as because the response to treatment selected resistant sub-population. The in vitro drug sensitivity assays demonstrated resistance to pyrimethamine, sulphadoxine and cycloguanil; while polymerase chain reaction (PCR) and restriction digest based methods indicated that at known drug resistant loci the isolate had a genotype of DHFR Val-16 and Thr-108 previously only associated with cycloguanil resistance. As per the published reports this type of paired mutations in natural isolates are rare. It is of considerable interest to carry out studies on alleles on alleles of this gene in relation to resistance at epidemiological level.     

Chloroquine-resistant malaria in Burma.

Two field trials to detect chloroquine-resistant malaria were conducted according to WHO recommendations in a malaria free area near Rangoon. Peripheral blood smears were examined for asexual forms of P. falciparum on day one through to day seven, on day 14, 21, and 28 after a standard dose of 1500 mg. of chloroquine base. Haskins test to detect chloroquine in urine was done on all cases and plasma chloroquine levels were measured in some. Out of 105 patients tested RI resistance was detected in 66, RII in 19 and RIII in three. Subsequent trials with other anti-malarial drugs indicated that the chloroquine-resistant P. falciparum were also resistant to one day therapy with pyrimethamine 50 mg. or sulphamethoxypyridazine 1 G given singly; and resistant to one day therapy with combinations of pyrimethamine 50 mg. plus sulphamethoxy pyridazine 1 G, pyrimethamine 13 mg. plus dapsone 100 mg., and trimethoprim 320 mg. plus sulphamethoxazole 1600 mg. All those tested were sensitive to quinine sulphate, 0-6 G given three times a day for 10 days, and were also sensitive to one day therapy with combinations of trimethoprim 500 mg. plus sulphalene 1 G, and pyrimethamine 50 mg. plus sulphamethoxine 1 G. Pyrimethamine 12-5 mg. plus dapsone 100 mg. in weekly doses was shown to be an effective chemoprophylaxis. Quinine was tested on 38 subjects while other drug schedule were tested on six to eight subjects.     

Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria.

Proguanil and pyrimethamine are antifolate drugs with distinct chemical structures that are used commonly in the prophylaxis and treatment of Plasmodium falciparum malaria. Clinical reports and field studies have suggested that some parasites refractory to proguanil can be treated with pyrimethamine, and vice versa. Analysis of the P. falciparum dihydrofolate reductase (DHFR) from different parasites reveals the structural basis for differential susceptibility to these antifolate drugs. Parasites harboring a pair of point mutations from Ala-16 to Val-16 and from Ser-108 to Thr-108 are resistant to cycloguanil (the active metabolite of proguanil) but not to pyrimethamine. A single Asn-108 mutation, on the other hand, confers resistance to pyrimethamine with only a moderate decrease in susceptibility to cycloguanil. Significant cross-resistance to both drugs occurs in parasites having mutations that include Ser-108----Asn-108 and Ile-164----Leu-164. These results reflect the distinct structures of pyrimethamine and cycloguanil and suggest fine differences in binding within the active site cavity of DHFR. Alternative inhibitors, used alone or in combination, may be effective against some strains of cycloguanil- or pyrimethamine-resistant malaria.     

Mechanisms of drug resistance in malaria

Plasmodium falciparum causes the most severe form of human malaria which directly results in over two million deaths per year. As there is not yet a useful vaccine against this disease the major form of treatment and control is the use of chemotherapeutic agents. Unfortunately the parasite has managed to devise mechanisms that allow it to evade the action of almost all the antimalarials in our arsenal. The antifolate drugs include the dihydrofolate inhibitors pyrimethamine and proguanil as well as the sulfones and sulfonamides. These antimalarials act on enzymes in the folate pathway. The mechanism of resistance to these compounds involve mutations in the target enzyme that decrease the affinity of binding of the drug. A second major group of antimalarials include the quinine-like compounds. Quinine was one of the first compounds used to treat malaria and the related drug chloroquine is the most important antimalarial. Mefloquine and halofantrine were developed in response to major problems with the spread of chloroquine resistance. Chloroquine resistance is due to the ability of the parasite to decrease the accumulation of the drug in the cell. The exact mechanism that allows this is still under investigation although at least one protein has been identified that affects the accumulation of this important antimalarial.     

Efficacies of chloroquine, pyrimethamine/sulphadoxine and pyrimethamine/sulphalene against P. falciparum in northeastern Nigeria.

The clinical and parasitologic efficacies of oral chloroquine phosphate, pyrimethamine/sulphadoxine and pyrimethamine/sulphalene in treating Plasmodium falciparum malaria were assessed in selected sites of northeastern Nigeria (Zone D of the Primary Health Care (PHC) Programme) using a 14-day standard in-vivo protocol during 1988-1990. Of a total of 2056 children under 5 years screened for infection, for chloroquine trials, 1189 (57.8%) were positive for Plasmodium infection. One hundred and seventy (14.3%) of these positive children were enrolled into the study. Clinically, the drug demonstrated high performance in clearing symptoms of infection. However, varying degrees of parasitologic failure, ranging from delayed clearance through recrudescence to asymptomatic Type-II resistance, were encountered. For tests with pyrimethamine/sulphadoxine and pyrimethamine/sulphalene, 517 and 253 children, respectively, were screened. The corresponding infection rates were 71.6% (370 children) and 71.5% (181 children), with 59 and 34 enrollments. Both drugs were highly effective, clinically and parasitologically. These findings and their implications for the success of the PHC programme for malaria control are discussed.     

Short communication: point mutations in the dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Colombia

Point mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum can lead to an increasing resistance of P. falciparum to pyrimethamine/sulphadoxine. We examined the prevalence of these mutations in 36 samples from Colombia. Analysed by polymerase chain reaction (PCR) for infection with P. falciparum, 25 (69%) tested positive. These positive isolates were tested further for point mutations in the genes of DHFR (codons 16, 51, 59, 108 and 164) and DHPS (codons 436, 437, 540, 581 and 613) by nested PCR and following mutation-specific restriction enzyme digestion. Gene mutations occurred in both the DHFR and DHPS gene of the Colombian isolates, suggesting that resistance to antifolate drugs exists or may develop soon in Colombia.     

Comparative efficacy of antimalarial drugs including ACTs in the treatment of uncomplicated malaria among children under 5 years in Ghana

The emergence and spread of Plasmodium falciparum resistance to commonly used antimalarials such as chloroquine and sulphadoxine/pyrimethamine poses major challenges to malaria control in sub-Saharan Africa. We undertook a study on the efficacy of some antimalarial drugs in 2003 with the view of supporting the National Malaria Control Programme in the review of the antimalarial drug treatment policy in Ghana. Children aged 6-59 months with signs/symptoms of uncomplicated malaria including axillary temperature > or =37.5 degrees C; mono infection with P. falciparum; and parent's willingness to give consent, were randomized into four treatment groups and followed up for a maximum of 28 days. The treatment groups were chloroquine (CHQ), sulphadoxine/pyrimethamine (SP), amodiaquine+artesunate (ADQ+ART) combination, and artemether+lumefantrine (Coartem) combination. Clinical evaluation of 168 children studied showed that cumulative pcr-corrected cure rates on day 28 were 100% for ADQ+ART; 97.5% for coartem, 60% for SP and 25% for CHQ. The artemisinin-based combinations effected rapid fever and parasite clearance. Prevalence of gametocytaemia was highest in the SP group whilst the CHQ group did not show any significant changes in haemoglobin levels during the follow-up period. The findings are in agreement with current recommendations for using artemisinin-based combinations for treating uncomplicated malaria in areas of high CHQ failure such as Ghana.     

Genetic evidence for Plasmodium falciparum resistance to chloroquine and pyrimethamine in Indochina and the Western Pacific between 1984 and 1998

Plasmodium falciparum resistance to chloroquine and pyrimethamine is widely distributed in malaria-endemic areas. The origin and geographic spread of this drug resistance have been inferred mainly from records of clinical resistance (treatment failure). Identification of the Plasmodium falciparum chloroqunie resistance transporter (pfcrt) gene and the dihydrofolate reductase (dhfr) gene as target genes of chloroquine and pyrimethamine, respectively, has made it possible to trace the history of genetic resistance to these two drugs. However, evidence for genetic resistance has been limited because of scarcity of archival specimens. We examined genotypes of pfcrt and dhfr in Indochina (Thailand, Myanmar, and Laos) and the Western Pacific (the Philippines, Indonesia, and Papua New Guinea) between 1984 and 1998 by testing samples obtained from malaria cases imported to Japan. Results show that 96% (28 of 29) and 77% (20 of 26) of samples had resistant genotypes of pfcrt and dhfr, respectively, substantiating the inferred history of clinical resistance in these geographic areas during this period.     

Polymorphisms in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes of Plasmodium falciparum and in vivo resistance to sulphadoxine/pyrimethamine in isolates from Tanzania

The efficacy of sulphadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of mutations associated with the active site sequence in the Plasmodium falciparum genes coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is associated with in vitro resistance to pyrimethamine and sulphadoxine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results show that alleles of DHPS (436-alanine, 437-alanine and 540-lysine) were significantly reduced in prevalence on day 7 after S/P treatment. In this area, a DHPS with 436-serine, 437-glycine and 540-glutamate appears to play a major role in resistance to S/P in vivo . Evidence for the influence of mutations in the DHFR gene in this investigation is not clear, probably because of the high prevalence of 'resistance-related' mutations at day 0 in the local parasite population. For apparently the same reason, it was not possible to show a statistical association between S/P resistance and the presence of particular polymorphisms in the DHFR and DHPS genes before treatment.     

Pyrimethamine and WR99210 exert opposing selection on dihydrofolate reductase from Plasmodium vivax

Plasmodium vivax is a major public health problem in Asia and South and Central America where it is most prevalent. Until very recently, the parasite has been effectively treated with chloroquine, but resistance to this drug has now been reported in several areas. Affordable alternative treatments for vivax malaria are urgently needed. Pyrimethamine-sulfadoxine is an inhibitor of dihydrofolate reductase (DHFR) that has been widely used to treat chloroquine-resistant Plasmodium falciparum malaria. DHFR inhibitors have not been considered for treatment of vivax malaria, because initial trials showed poor efficacy against P. vivax. P. vivax cannot be grown in culture; the reason for its resistance to DHFR inhibitors is unknown. We show that, like P. falciparum, point mutations in the dhfr gene can cause resistance to pyrimethamine in P. vivax. WR99210 is a novel inhibitor of DHFR, effective even against the most pyrimethamine-resistant P. falciparum strains. We have found that it is also an extremely effective inhibitor of the P. vivax DHFR, and mutations that confer high-level resistance to pyrimethamine render the P. vivax enzyme exquisitely sensitive to WR99210. These data suggest that pyrimethamine and WR99210 would exert opposing selective forces on the P. vivax population. If used in combination, these two drugs could greatly slow the selection of parasites resistant to both drugs. If that is the case, this novel class of DHFR inhibitors could provide effective and affordable treatment for chloroquine- and pyrimethamine-resistant vivax and falciparum malaria for many years to come.     

Short communication: Prevalence of mutations associated with resistance to atovaquone and to the antifolate effect of proguanil in Plasmodium falciparum isolates from northern Ghana

Atovaquone-proguanil has recently been introduced for the treatment and prophylaxis of malaria. However, resistance of Plasmodium falciparum is increasingly reported. We assessed P. falciparum polymorphisms associated with resistance to atovaquone (cytochrome b, cytb) and to cycloguanil, the active compound of proguanil (dihydrofolate reductase, dhfr) in 100 isolates from northern Ghana. None of these exhibited cytb codon 268 mutations. Moreover, no dhfr V16A, S108T or I164L mutations linked with cycloguanil resistance were detected. However, dhfr triple mutants (S108N-I51L-C59R) conferring resistance to proguanil and sulphadoxine-pyrimethamine were seen in 51% of the isolates. In northern Ghana, P. falciparum cytb codon 268 mutations associated with atovaquone resistance are absent. Although proguanil appears to act synergistically with atovaquone in a way different from its antifolate property, the abundance of dhfr polymorphisms will likely compromise the prevention of dissemination of atovaquone-resistant parasites once emerged.     

Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil

Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatment and prevention of chloroquine-resistant Plasmodium falciparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that point mutations at key positions of the dihydrofolate reductase-thymidylate synthase gene are strongly associated with antifolate resistance. However, whether the same or distinct mutations are involved in the development of resistance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. falciparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase domain of the gene (from 34 of 42 isolates) to analyze the genotype that may distinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n = 11 isolates) was associated with low 50% inhibitory concentrations (IC50s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.60-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid substitution, Asn-108. Only three mutant codons were observed among the antifolate-resistant isolates: Ile-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC50 and, to a much lesser extent, cycloguanil IC50. These results support a partial cross-resistance between pyrimethamine and cycloguanil that is based on similar amino acid substitutions in dihydrofolate reductase and suggest that two or three mutations, including at least Asn-108, may be necessary for cycloguanil resistance, whereas a single Asn-108 mutation is sufficient for pyrimethamine resistance.     

Plasmodium falciparum pfcrt and pfmdr1 polymorphisms are associated with the pfdhfr N108 pyrimethamine-resistance mutation in isolates from Ghana

The Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) T76 and multidrug resistance gene analogue (pfmdr1) Y86 mutations are associated with chloroquine(CQ)-resistance. In isolates from 172 pregnant women living in the area of Agogo, Ghana, pfcrt T76 was detected in 69% and pfmdr1 Y86 in 66%. Pfcrt T76 but not pfmdr1 Y86 was more prevalent in samples from women with residual CQ in urine or serum. Parasite densities and multiplicity of infection of pfmdr wild type but not of resistant isolates were reduced by CQ. Adjusted for CQ and pyrimethamine (PYR) in urine, the P. falciparum dihydrofolate reductase (pfdhfr) N108 mutation which confers PYR-resistance was 3.1 and 3 times, respectively, more likely to be detected in isolates containing pfcrt and pfmdr1 mutations than in those comprising wild type alleles. Pfcrt, pfmdr, and pfdhfr mutations are frequent in P. falciparum from this part of Ghana which may limit the choice of drugs for the prevention of malaria in pregnancy. The association of CQ- and PYR-resistance mutations independent of recent drug use could indicate accelerated development of resistance to structurally unrelated drugs. Alternatively, it may reflect selection of resistance in persisting infections due to no longer detectable drug pressure.     

Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria.

Analysis of a genetic cross of Plasmodium falciparum and of independent parasite isolates from Southeast Asia, Africa, and South America indicates that resistance to pyrimethamine, an antifolate used in the treatment of malaria, results from point mutations in the gene encoding dihydrofolate reductase-thymidylate synthase (EC 1.5.1.3 and EC 2.1.1.45, respectively). Parasites having a mutation from Thr-108/Ser-108 to Asn-108 in DHFR-TS are resistant to the drug. The Asn-108 mutation occurs in a region analogous to the C alpha-helix bordering the active site cavity of bacterial, avian, and mammalian enzymes. Additional point mutations (Asn-51 to Ile-51 and Cys-59 to Arg-59) are associated with increased pyrimethamine resistance and also occur at sites expected to border the active site cavity. Analogies with known inhibitor/enzyme structures from other organisms suggest that the point mutations occur where pyrimethamine contacts the enzyme and may act by inhibiting binding of the drug.     

Chloroquine resistance of Plasmodium falciparum malaria in Ethiopia and Eritrea

We report on the first 2 years of operation of a new strategy for treatment for P. falciparum malaria patients who were not cured by a standard course of chloroquine. Any such patient who returned to a malaria treatment and detection post within 2 weeks was treated daily under supervision with chloroquine. Patients whose parasitaemia had not decreased below 25% of the initial level by day 3 or cleared completely by day 7 were given sulphadoxine/pyrimethamine (Fansidar). Of 39 824 patients treated initially with chloroquine, 4% returned to the malaria post within 2 weeks of treatment; 87% of these were chloroquine resistant and treated with Fansidar and 28% of the returning patients were RIII resistant. Resistance was associated with geographical area, initial parasite density and age. Earlier studies had shown resistance to be confined to border areas, but we found that it was highest in the centre of the region, notably in the lowlands of the Shewa and Arsi provinces, and lowest in the west. Although imported cases have been held responsible for the development of resistance in border areas, other factors are likely to be important in the middle of the region. The implications of these findings for a treatment policy of P. falciparum malaria in the region are discussed.     

Urban malaria in Dakar, Senegal: chemosusceptibility and genetic diversity of Plasmodium falciparum isolates

The chemosusceptibility and genetic polymorphism of Plasmodium falciparum populations from 48 patients hospitalized for malaria at the Hospital Principal in Dakar, Senegal were investigated during the 2002 malaria transmission season. Sixty-two percent of the isolates collected were from patients with severe malaria and 38% were from patients with mild malaria. In vitro activities of chloroquine, quinine, cycloguanil, atovaquone, mefloquine, halofantrine, and artesunate were evaluated. The prevalence of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) and dihyropteroate synthetase (dhps) genes and the P. falciparum chloroquine resistance transporter (Pfcrt) gene associated with cycloguanil, pyrimethamine, sulfadoxine, and chloroquine resistance were estimated. The genetic polymorphism of the parasite populations was evaluated by analysis of the highly polymorphic regions of merozoite surface protein 1 (msp1) block 2 and msp2. Seventy percent of the isolates were assessed by an in vitro assay. Fifty-two percent of the isolates were chloroquine resistant, 45% were cycloguanil resistant, and 24% were atovaquone resistant. Four percent had low susceptibility to quinine. The Pfcrt and dhfr mutations were associated with in vitro chloroquine- and antimetabolic drug-resistant isolates, respectively. Approximately 70% of the isolates contained two or more clones. Genetic diversity of P. falciparum was high. The prevalence of allelic family K1 of msp1 was 68%. Isolates of P. falciparum were highly resistant to chloroquine, cycloguanil and atovaquone. The transmission rate of malaria in Dakar is low but a high degree of genetic polymorphism can increase severe malaria, as shown by persons coming to Dakar from areas highly endemic for malaria. Areas with urban malaria should use vector control measures and efficient chemoprophylaxis for non-immune populations.     

Epidemiology of drug-resistant malaria in Republic of Congo: using molecular evidence for monitoring antimalarial drug resistance combined with assessment of antimalarial drug use

In Congo, urgent efforts are needed to help with the revision of the national antimalarial drug policy. Despite its high resistance level, chloroquine (CQ) is still extensively used as the first-line treatment for uncomplicated Plasmodium falciparum malaria. The study was conducted in children under 5 years with uncomplicated malaria in Pointe-Noire and Brazzaville, the two largest cities that contain approximately 60% of the population of Congo. We investigated by polymerized chain reaction and sequencing methods the frequency distribution of molecular markers for antimalarial drug resistance, including mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene associated with CQ resistance and mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes conferring resistance to sulphadoxine/pyrimethamine (SP) among pre-treatment P. falciparum isolates, as well as assessing antimalarial drug use in the community. pfcrt (K76T) mutation was present in most isolates (96.4%, n = 138) and high frequency (69.2%, n = 133) of triple-mutant dhfr-S108N, N51I, C59R was observed. The quintuple mutant (dhfr-S108N, N51I, C59R and dhps-A437G or S436A, K540E) considered as molecular marker for SP treatment failure was not found because dhps-K540E mutation was absent in isolates tested; this is a clear evidence for the excellent efficacy of SP that we previously described in the same population. The complete absence of the dhps-K540E mutation is a deterrent component for using this molecular marker as an early warning tool for SP resistance testing in that population. Poor compliance issues related to the antimalarial drug use including inappropriate manufacturing practices reported in this study require intensive attention and should be taken into account when implementing drug policy change. If Congo changes its treatment policy from CQ to SP monotherapy, this will not last long. The strategy of combining SP with other affordable and effective antimalarial drugs such as the artemisinin derivatives to improve efficacy and to delay the development of parasite resistance is essential.     

Drug resistance and genetic diversity of Plasmodium falciparum parasites from suriname

Plasmodium falciparum in Suriname was studied for the presence of drug resistance and genetic variation in blood samples of 86 patients with symptomatic malaria. Drug resistance was predicted by determining point mutations in the chloroquine resistance marker of the P. falciparum chloroquine resistance transporter (pfcrt) gene (codon 76) and the pyrimethamine-sulfadoxine resistance markers in the dihydrofolate reductase (dhfr) gene (codons 16, 51, 59, 108, and 164) and dihydropteroate synthase (dhps) gene (codons 436, 437, 540, 581, and 613). Genetic variability was determined by sequence analysis of the polymorphic segments of the merozoite surface protein 2 (msp-2) and glutamate-rich protein (glurp) genes. Mutations in the pfcrt, dhps, and dhfr genes were found in all samples tested, suggesting that resistance to chloroquine and antifolate drugs is present at a high frequency. A low number of alleles was found for the msp-2 and glurp genes. This indicates limited genetic diversity and, based on geographic data, a genetically homogeneous P. falciparum population in Suriname.     

Prevalence of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum isolates from southern Mauritania

The increasing resistance of Plasmodium falciparum in the treatment of uncomplicated malaria with pyrimethamine/sulphadoxine has been associated in several studies with the occurrence of point mutations in the genes of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). In this study, the prevalence of these mutations was examined in samples from south-east Mauritania, where atypically strong rainfalls in 1998 and 1999 led to a severe outbreak of falciparum malaria. We analysed 386 samples by polymerase chain reaction (PCR) for infection with P. falciparum, of which 162 (41.97%) were positive. These isolates were examined for point mutations in the genes of DHFR (codons 16, 51, 59, 108 and 164) and DHPS (codons 436, 437, 540, 581 and 613) by nested PCR and subsequent mutation-specific restriction enzyme digest. We found a low overall prevalence of DHFR gene mutations (up to 18.6% of isolates), but a high overall prevalence of DHPS gene mutations (up to 49.1% of isolates). Thus, emerging resistance to antifolate drugs may be expected to develop soon in the investigated area. This study demonstrates the utility of simple, relatively rapid and inexpensive molecular methods and their application in surveillance programmes. Testing for prevalence of point mutations conferring antifolate resistance might help to identify the developing of drug resistance at a very early stage.     

Drug resistance in Plasmodium falciparum from the Chittagong Hill Tracts, Bangladesh

OBJECTIVE: To assess the efficacy of antimalarial treatment and molecular markers of Plasmodium falciparum resistance in the Chittagong Hill Tracts of Bangladesh. METHODS: A total of 203 patients infected with P. falciparum were treated with quinine 3 days plus sulphadoxine/pyrimethamine (SP) combination therapy, and followed up during a 4-week period. Blood samples collected before treatment were genotyped for parasite mutations related to chloroquine (pfcrt and pfmdr1 genes) or SP resistance (dhfr and dhps). RESULTS: Of 186 patients who completed follow-up, 32 patients (17.2%) failed to clear parasitaemia or became positive again within 28 days after treatment. Recurring parasitaemia was related to age (chi(2) = 4.8, P < 0.05) and parasite rates on admission (t = 3.1, P < 0.01). PCR analysis showed that some of these cases were novel infections. The adjusted recrudescence rate was 12.9% (95% CI 8.1-17.7) overall, and 16.6% (95% CI 3.5-29.7), 15.5% (95% CI 8.3-22.7) and 6.9% (95% CI 0.4-13.4) in three age groups (<5 years, 5-14, > or =15). The majority of infections carried mutations associated with chloroquine resistance: 94% at pfcrt and 70% at pfmdr. Sp-resistant genotypes were also frequent: 99% and 73% of parasites carried two or more mutations at dhfr and dhps, respectively. The frequency of alleles at dhfr, dhps and pfmdr was similar in cases that were successfully treated and those that recrudesced. CONCLUSIONS: The clinical trial showed that quinine 3-days combined to SP is still relatively effective in the Chittagong Hill Tracts. However, if this regimen is continued to be widely used, further development of SP resistance and reduced quinine sensitivity are to be expected. The genotyping results suggest that neither chloroquine nor SP can be considered a reliable treatment for P. falciparum malaria any longer in this area of Bangladesh.