In vivo chloroquine resistance and prevalence of the pfcrt codon 76 mutation in Plasmodium falciparum isolates from the Republic of Congo

Chloroquine (CQ) resistance in Plasmodium falciparum has been particularly associated with mutations in the pfcrt gene. The present study was carried out in the malaria hyperendemic town of Brazzaville (Republic of Congo, Central Africa) where CQ is still recommended and used as a first-line drug for P. falciparum malaria. We assessed the efficacy of CQ in vivo, and the association between pfcrt mutation at codon 76 and treatment outcome in 50 children with uncomplicated malaria. The failure rate on day 28 was 95.7% and the pfcrt K76T mutation was present in 100% of isolates. No variation in the multiplicity of infection was observed in pre- and post-treatment isolates. In further 87 isolates from uncomplicated patients not treated with CQ, the mutation was detected in 98.5% of isolates. This study confirms the high level of in vivo resistance to CQ and shows the high prevalence of pfcrt K76T mutation in the Republic of Congo.     

Chloroquine-treatment failure in northern Ghana: roles of pfcrt T76 and pfmdr1 Y86

Although chloroquine (CQ) monotherapy is now generally inadequate for the treatment of Plasmodium falciparum malaria in northern Ghana--recently, 58% of 225 children failed treatment by day 14--use of the drug continues because of its low cost and wide availability. The risk factors associated with CQ-treatment failure in this region of Africa, including the T76 mutation in the chloroquine resistance transporter (pfcrt) gene and the Y86 mutation in the multidrug resistance (pfmdr1) gene of P. falciparum, have now been investigated, and genotype-failure indices (GFI) have been calculated.Treatment failure was found to be associated with young age, poor nutritional status, pfcrt T76 and pfmdr1 Y86, and early treatment failure (ETF) was also associated with high parasitaemia. The presence and concentration of 'residual' CQ in the blood of patients immediately before they were treated with CQ for the present study appeared to have no effect on outcome. Presence at recruitment of pfcrt T76 or pfmdr1 Y86 or both mutations increased the risk of treatment failure by 3.2-, 2.4- and 4.5-fold, and the risk of ETF by 9.8-, 2.7- and 10.2-fold, respectively. The pfcrt T76 GFI for clinical and all treatment failures were 2.8 and 1.4, respectively. These indices were relatively low in the younger children, those with malnutrition, and those with high parasitaemias when treated. Residual CQ did not affect the GFI substantially.Both pfcrt T76 and, to a lesser extent, pfmdr1 Y86 would be useful tools for the surveillance of CQ resistance in northern Ghana. In the current transition phase to alternative first-line treatment for P. falciparum malaria, it should be possible to provide estimates of the level of CQ resistance by monitoring the prevalences of these mutations.     

Association of pfcrt but not pfmdr1 alleles with chloroquine resistance in Iranian isolates of Plasmodium falciparum

This study was designed to analyze the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) mutations as markers of chloroquine (CQ) resistance in 200 blood samples collected from malaria patients in south-eastern Iran during 2002-2005. Among these, 25 (post-treatment) fulfilled the 28-day follow-up study. A high number of Iranian P. falciparum (97%) strains harbored quadruple mutations at codons 76T, 220S, 326D, and 356L. All post-treatment isolates harbored the mutant allele 76T, but low rates of the mutant allele 86Y (44%) of the pfmdr1 gene were detected. No wild haplotype of pfcrt (72-CVMNKAQNIR-371) was found in post-treatment samples; however, 56% of clinical "failure" samples carried the wild type of pfmdr1 (NYSND). The present results suggest a strong association between pfcrt 76T, but not pfmdr1 86Y mutation and in vivo CQ resistance. Furthermore, we found the CQ resistance-associated SVMNT haplotype, which previously had been seen in South American isolates. Although Iran is located more proximally to Southeast Asia than to South America, no CQ resistance-associated CVIET haplotye has been observed in this region. Therefore, these results were not consistent with the earlier presumed spread of CQR parasites from Southeast Asia to Africa via the Indian subcontinent. In conclusion, P. falciparum mutations associated with resistance to CQ are abundant in south-eastern Iran and this finding strongly supports that CQ as the first line drug is inadequate for treatment of uncomplicated falciparum malaria in Iran.     

Association of Plasmodium falciparum chloroquine resistance transporter variant T76 with age-related plasma chloroquine levels

The mutation leading to the substitution of a threonine (T76) for a lysine at position 76 of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was genotyped in 100 Nigerian children with asymptomatic parasitemia. Isolates containing both pfcrt variants were found to harbor higher numbers of parasite clones (P < 0.002). The prevalence of the pfcrt T76 variant decreased with age (P < 0.0001) and increased with blood levels of chloroquine (CQ) (P < 0.0001). Whereas the K76 allele was more frequent in individuals without detectable plasma CQ levels (79.7%), only the pfcrt T76 variant was observed in children with CQ levels > 150 nmol/L. In individuals without detectable plasma CQ, the proportion of those with pfcrt T76 decreased from 60% in children < 2 years old to 23.5% in children > or = 6 years old (P < 0.01). The association of actual blood levels of CQ and the occurrence of pfcrt T76 underlines that the pfcrt T76 variant is in fact the mediator of CQ resistance.     

High prevalence of pfcrt K76t mutants among Plasmodium falciparum isolates from Sabah, Malaysia

Chloroquine (CQ) remains the first line drug for the prevention and treatment of malaria in Malaysia in spite of the fact that resistance to CQ has been observed in Malaysia since the 1960s. CQ-resistance is associated with various mutations in pfcrt, which encodes a putative transporter located in the digestive vacuolar membrane of P. falciparum. Substitution of lysine (K) to threonine (T) at amino acid 76 (K76T) in pfcrt is the primary genetic marker conferring resistance to CQ. To determine the presence of T76 mutation in pfcrt from selected areas of Kalabakan, Malaysia 619 blood samples were screened for P. falciparum, out of which 31 were positive. Blood samples were collected on 3 MM Whatman filter papers and DNA was extracted using QIAmp DNA mini kit. RFLP-PCR for the detection of the CQ-resistant T76 and sensitive K76 genotype was carried out. Twenty-five samples were shown to have the point mutation in pfcrt whereas the remaining samples were classified as CQ-sensitive (wild-type). In view of the fact that CQ is the first line anti-malarial drug in Malaysia, this finding could be an important indication that treatment with CQ may no longer be effective in the future.     

Prevalence of the K76T mutation in the putative Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene and its relation to chloroquine resistance in Mozambique

K76T, a mutation in the Plasmodium falciparum chloroquine (CQ) resistance transporter protein, has been implicated in resistance to CQ. A modified 14-day in vivo test to estimate the CQ resistance level was done in southern Mozambique: 21 (42%) of 50 subjects who completed the follow-up were CQ susceptible. Use of msa2-restriction fragment length polymorphism (RFLP) genotyping to differentiate new from recrudescent infections made little difference in the estimated prevalence of resistance. The K76T mutation prevalence was estimated by RFLP-polymerase chain reaction and sequencing, and its relation to parasitological CQ resistance was explored on day 0 samples: 51 of 56 pretreatment samples presented the T76 codon, and it was present in 100% of children with parasitological resistance. T76 also was present in 18 of 23 subjects in whom the infection resolved after CQ treatment. These findings show a high prevalence of the K76T mutation among wild isolates but also suggest additional factors responsible for CQ resistance.     

Quinoline resistance associated polymorphisms in the pfcrt, pfmdr1 and pfmrp genes of Plasmodium falciparum in Iran

Plasmodium falciparum resistance to chloroquine (CQ) has been documented in Iran since the early 1980s and has since gradually increased. Iran is therefore reviewing its national drug policy for malaria control. We describe the prevalence of single nucleotide polymorphisms (SNP) associated with quinoline drug resistance in south eastern Iran. Pre-treatment blood from patients with uncomplicated but symptomatic P. falciparum infection was analysed. Polymorphisms at codons 76, 152, 163 and 220 of the pfcrt gene (chloroquine resistance transporter) and at codons 86, 184, 1034, 1042 and 1246 of the pfmdr1 gene (multidrug resistance) were determined by PCR-RFLP and sequencing. In addition, SNPs on a recently described multidrug resistance protein (pfmrp) and a microsatellite (MS-4760) in the pfnhe-1 (sodium hydrogen exchanger) gene associated with quinoline and quinine resistance, respectively, were investigated for the first time in field samples not from Thailand. pfcrt 76T was found in 99% and pfmdr1 86Y in 72% of the samples. pfmrp 191H and 437S associated with decreased quinoline response were found to be absolutely linked at a frequency of 13.6%. The pfnhe-1 MS-4760 one repeat allele associated to quinine response in vitro was also detected. Sequencing of the pfcrt 72-76 haplotype revealed that SVMNT was the most common allele as previously observed in India. This suggests that pfcrt found in the Iranian P. falciparum population may have the same origin as in the P. falciparum populations in India but different from that normally found in south east Asia. In conclusion, the frequencies of quinoline resistance associated gene polymorphisms in this region suggest a population that has been significantly selected for by the long use of CQ.     

Polymorphism of the Plasmodium falciparum multidrug resistance and chloroquine resistance transporter genes and in vitro susceptibility to aminoquinolines in isolates from the Peruvian Amazon

In vitro drug sensitivity to chloroquine (CQ), mefloquine (MQ) and quinine was investigated in 60 culture-adapted Plasmodium falciparum isolates from malaria patients in Padrecocha, a village in the Amazonian Department of Loreto, Peru. All isolates showed resistance to CQ, decreased susceptibility to quinine, and sensitivity to MQ. These isolates were examined for mutations in the P. falciparum multidrug resistance 1 (pfmdr1) and chloroquine resistance transporter (pfcrt) genes previously linked to CQ resistance. The mutations N86Y and D1246Y, two of the five mutations commonly observed in the pfmdr1 gene of CQ-resistant clones, were not found. The pfcrt mutation K76T, associated with CQ resistance, was identified in all the isolates tested. Sequence analysis of codons 72-76 in the pfcrt gene showed the haplotypes SVMNT and CVMNT.     

Efficacy of chloroquine in the treatment of uncomplicated,Plasmodium falciparum malaria in northern Ghana

Chloroquine (CQ) resistance in Plasmodium falciparum contributes to growing malaria-attributable morbidity and mortality in sub-Saharan Africa. However, the extent and degree of such resistance vary considerably between endemic areas. Data on CQ resistance in northern Ghana are almost entirely lacking. The therapeutic efficacy of CQ in uncomplicated malaria was therefore assessed, in a standard, 14-day protocol, in 225 children aged <5 years in Tamale, in the Northern region of Ghana. Early treatment failure (ETF) was observed in 11% of the children and late treatment failure in 18%. High initial parasite density and young age were independent predictors for ETF. Resistant parasitological responses (RI-RIII) were seen in 57% of the cases that could be classified. More than half of these responses occurred in children fulfilling the criteria for adequate clinical response (ACR), indicating a considerable lack of agreement between parasitological and clinical outcome. During the follow-up period, haemoglobin levels increased by approximately 1g/dl not only in patients with ACR but also in those who experienced clinical failure more than 1 week post-treatment. As CQ-treatment failure occurred in >25% of the children and more than half of the parasitological responses indicated resistance, current recommendations for the treatment of uncomplicated malaria in young children in northern Ghana have to be reconsidered.     

Comparison of the therapeutic efficacy of chloroquine and sulphadoxine-pyremethamine in children and pregnant women

Objective To compare the parasitological failure rates of under‐fives and pregnant women with parasitaemia treated with chloroquine (CQ) or sulphadoxine‐pyrimethamine (SP). Methods During a clinical trial of CQ, SP, amodiaquine (AQ) and SP plus AQ combination for malaria treatment in pregnant women in Ghana, a parallel study of treatment of children below 5 years of age with symptomatic malaria with CQ and SP was undertaken. Four hundred and fifty pregnant women with malaria parasitaemia and 203 children with malaria parasitaemia were randomized to receive CQ or SP. They were followed up and parasitological failure by days 14 and 28 after the start of treatment was assessed. Results Polymerase chain reaction (PCR)‐uncorrected parasitological failure rates by day 28 after the start of treatment with CQ were 58.5% (55/94), 38.5% (45/117), 31% (13/42) and 8.2% (4/49) in children, primigravidae, secundigravidae and multigravidae, respectively. For those treated with SP the rates by day 28 were 36.4% (32/88), 27.1% (29/107), 6.1% (3/49) and 3.8% (2/52) in children, primigravidae, secundigravidae and multigravidae, respectively. In both CQ and SP treatment arms, children were twice as likely to experience recrudescence as pregnant women (RR 2.1 [95% CI 1.6–2.6] P < 0.0001) by day 28 after the start of treatment. Conclusions Parasitological failure rates were significantly lower in asymptomatic pregnant women, particularly in multigravidae, compared with symptomatic children. Reliance on drug sensitivity results observed in children only to decide on antimalarial regimes for pregnant women may not be appropriate.     

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.     

The T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia.

The T76 mutation in the pfcrt gene has been linked to chloroquine (CQ) resistance in Plasmodium falciparum. PCR-based analysis of pfcrt alleles was performed on pre-treatment samples from 107 individuals who had P. falciparum infections and lived in Papua, Indonesia. The results of a 28-day, in-vivo test revealed clinical resistance to CQ in 79 (74%) of the samples. The crude sensitivity of the pfcrt T76 assay for detecting the CQ-resistant infections in the samples was 96% and the crude specificity 52%. Discordance between pfcrt genotype and in-vivo phenotype was analysed either by genotyping of the merozoite surface protein-2 (to distinguish re-infection from recrudescence) or by amplification of the P. falciparum-specific small-subunit ribosomal RNA (ssrRNA) gene, using nested PCR (to detect any sub-patent but resistant parasites in infections misclassified as sensitive by the in-vivo test). When adjusting for the results of these analyses, the sensitivity and specificity of the pfcrt T76 assay for detecting the CQ-resistant infections became 93% and 82%, respectively. Overall, the present results indicate that the pfcrt T76 assay may be used to forecast therapeutic failure caused by CQ resistance. Validation requires exploration of the phenotype classifications based on the results of in-vivo tests, using genetic analyses that distinguish re-infection from recrudescence and detect microscopically subpatent parasitaemias.     

Improved efficacy with amodiaquine instead of chloroquine in sulfadoxine/pyrimethamine combination treatment of falciparum malaria in Uganda: experience with fixed-dose formulation

Amodiaquine (AQ) is an affordable compound, chemically related to chloroquine (CQ) but often effective against CQ resistant Plasmodium falciparum. In Uganda, a pre-packed fixed-dose combination of CQ plus sulfadoxine/pyrimethamine (CQ+SP) called Homapak is used in the home based management of fever program (HBM). We performed a single blind randomized trial to determine the efficacy of AQ+SP in comparison with the fixed-dose CQ+SP (Homapak) in the treatment of uncomplicated falciparum malaria in Ugandan children aged 6 months to 5 years. The study was done in 2004 at Walkuba Health Center, a sub-urban area in Jinja district, Uganda. Primary outcome was the day 14 per protocol clinical and parasitological response according to the WHO. A total of 183 children were included (mean age 28 months) and 90% completed 28 days of follow up. The day 14 adequate clinical and parasitological response was 70.9% for CQ+SP and 97.4% for AQ+SP (p<0.001). In those given CQ+SP, treatment failure rates for the 6 months to 2 years age group were much higher (48.2%) than in the older children (18.2%, p=0.004). The day 28 PCR adjusted parasitological failure rates were also higher in the CQ+SP (31.3%) than in the AQ+SP group (13.1%) (p=0.003), with a higher gametocyte carriage among the CQ+SP group. We conclude that the efficacy of AQ+SP was significantly superior to the fixed-dose CQ+SP (Homapak), particularly among the youngest children. Thus, AQ could be used instead of CQ in combination with SP to improve the effectiveness against falciparum malaria in Uganda.     

Chloroquine and sulphadoxine-pyrimethamine efficacy for uncomplicated malaria treatment and haematological recovery in children in Bobo-Dioulasso,Burkina Faso during a 3-year period 1998-2000

We determined the parasitological resistance and the clinical failure to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) by the WHO 14-day in vivo test over three consecutive years in 948 children aged 6-59 months with uncomplicated malaria attending four health centres in the province of Houet, Burkina Faso. Children were alternatively allocated to either CQ or SP. Packed cell volume (PCV) was measured at days 0 and 14. Parasitological resistance (RI, RII and RIII) to CQ was 18% (83 of 455) and to SP <1% (two of 308). Clinical failure with CQ was 12% (53 of 455) with no evidence of increase over time. Only one case of clinical failure was detected among the children treated with SP. The prevalence of anaemia (PCV <25%) was about 40% at day 0 and had decreased substantially by day 14 in both groups. However, in children treated with SP the prevalence of anaemia at day 14 was significantly lower than in those treated with CQ:RR = 3.15 (95% CI: 1.33-7.42, P = 0.008). CQ and SP are still efficacious for the treatment of uncomplicated malaria in children, at least in this area of Burkina Faso. However, the prevalences of CQ resistance reported from other areas of the country are worrying because of its potential spread. Regular surveillance of resistance to commonly used antimalarial drugs should continue.     

Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China

Resistance of Plasmodium falciparum to chloroquine (CQ) is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene and modified by other mutations in this gene and in the P. falciparum multidrug resistance 1 (pfmdr1) gene. To determine the extent of polymorphisms in these genes in field P. falciparum isolates from Yunnan province of China, we genotyped the pfcrt codon 76, pfmdr1 codons 86 and 1246. Our results showed that although CQ has been withdrawn from treating falciparum malaria for over two decades, 90.3% of the parasites still carried the pfcrt K76T mutation. In contrast, mutations at pfmdr1 codons 86 and 1246 were rare. Sequencing analysis of the pfcrt gene in 34 parasite field isolates revealed CVIET at positions 72-76 as the major type, consistent with the theory of Southeast Asian origin of CQ resistance in the parasite. In addition, two novel pfcrt haplotypes (75D/144Y/220A and 75E/144Y/220A) were identified. Real-time polymerase chain reaction was used to determine pfmdr1 gene amplification, which is associated with mefloquine resistance. Our result indicated that in agreement with that mefloquine has not been used in this area, most (>90%) of the parasites had one pfmdr1 copy. Genotyping at two hypervariable loci showed relatively low levels of genetic diversity of the parasite population. Meanwhile, 28.4% of cases were found to contain mixed clones, which favour genetic recombination. Furthermore, despite a unique history of antimalarial drugs in Yunnan, its geographical connections with three malarious countries facilitate gene flow among parasite populations and evolution of novel drug-resistant genotypes. Therefore, continuous surveillance of drug resistance in this area is necessary for timely adjustment of local drug policies and more effective malaria control.     

Point mutations in the pfcrt and pfmdr-1 genes of Plasmodium falciparum and clinical response to chloroquine,among malaria patients from Nigeria

Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with specific point mutations in the pfcrt and pfmdr-1 genes. In the present study, 30 children aged 1-12 years, who were all suffering from acute, uncomplicated, P. falciparum malaria in Ibadan, Nigeria, were evaluated to assess the association between these mutations and clinical outcome following treatment with CQ. The parasites, in blood samples collected pre-treatment and, in those who failed treatment, on the day symptoms re-occurred post-treatment, were genotyped using the polymorphic MSP1, MSP2 and GLURP loci and PCR-RFLP. The results showed that, pre-treatment, all 30 patients had polyclonal infections, the mean numbers of P. falciparum clones detected per infection being 2.6 with MSP1, 4.2 with MSP2 and 2.8 with GLURP. The T76 allele of pfcrt and the Y86 allele of pfmdr-1 were found in 53% and 40%, respectively, of the pre-treatment samples from the 15 patients who failed CQ treatment, but the Y1246 mutation in pfmdr-1 was never detected. Although the parasites from the two patients with high-grade (RIII) resistance to CQ had both of these point mutations, the presence of the T76 allele of pfcrt or the Y86 allele of pfmdr-1 (considered individually) could not be used to predict treatment outcome. However, a high frequency of clonal multiplicity may confound attempts to associate the point mutations in pfcrt or pfmdr-1 with clinical response to CQ. It remains unclear whether the present results represent the characteristics of the predominant parasite populations in the study area. Further studies are needed before the strength of the association between the point mutations identified as markers of drug resistance and clinical outcome can be accurately evaluated, in this and other regions of intense transmission.     

Molecular markers for chloroquine-resistant Plasmodium falciparum malaria in Thailand and Laos.

Chloroquine-resistant Plasmodium falciparum is well documented in Thailand. Laos, however, continues to use chloroquine (CQ) as the first-line therapy for the treatment of P. falciparum malaria. The objective of the present study was to determine the prevalence, in these two areas, of the cg2, pfmdr1 and pfcrt allelic types that have previously been associated with CQ resistance. Isolates of P. falciparum were collected from participants in ongoing treatment studies conducted in Thailand (near the Thai-Cambodian border) and in Laos (Vang Vieng district). The pfmdr1 and pfcrt alleles were characterized by PCR-RFLP and mutations in cg2 were characterized by PCR and single-stranded-conformation-polymorphism (SSCP) electrophoresis. Eight (32%) of the 25 Laotian isolates but only one (4%) of the 25 Thai isolates were found to contain the pfmdr1 mutation N86Y (P = 0.02). In contrast, the cg2 polymorphisms previously associated with CQ resistance were present in only 10 of the isolates from Laos but 24 of those from Thailand (40% v. 96%; P < 0.001). All the samples from both countries contained the pfcrt K76T mutant allele reported to confer resistance to CQ. The results may indicate that drug pressure for the maintenance of the pfmdr1 and cg2 alleles varies in intensity in the Thai and Laotian study areas, probably reflecting differences in the national malaria-treatment policies of Thailand and Laos.     

云南边境地区氯喹及与青蒿琥酯伍用治疗前后恶性疟原虫pfcrt 和pfmdr1抗药性有关基因点突变的变化特征

目的　了解氯喹单用及与青蒿琥脂伍用治疗恶性疟前后 ,pfcrt和 pfmdr1抗药性有关基因的点突变变化特征。　 方法　使用PCR RFLP技术检测基因点突变。　结果　氯喹及与青蒿琥脂伍用治疗前后的所有样本都发现有恶性疟原虫pfcrt基因氨基酸编码 76突变为苏氨酸的特征。但是 ,氯喹治疗前 ,5 0 % pfmdr1基因氨基酸编码 86为天冬酰氨酸 (野生型 ) ,而剩余的 5 0 %为野生型和突变型 (苏氨酸 )的特征。氯喹治疗后 ,在 18个复燃的病例中 ,83 .3 %的 pfmdr1基因 86位点为野生型 ,剩余的 16.7%是混合型。氯喹与青蒿琥脂伍用治疗前 ,3个样本携带混合型基因型 ,剩余的 (86% )为野生型 ,但治疗后 ,所有样本只携带野生型。　结论　这些结果可能支持这样的假说 :pfcrt基因突变起主导作用 ,但 pfmdr1基因突变增强了氯喹抗药性的效果。     

Low efficacy of amodiaquine or chloroquine plus sulfadoxine-pyrimethamine against Plasmodium falciparum and P. vivax malaria in Papua New Guinea

Because of increasing resistance to 4-aminoquinolines in Papua New Guinea, combination therapy of amodiaquine (AQ) or chloroquine (CQ) plus sulfadoxine-pyrimethamine (SP) was introduced as first-line treatment against uncomplicated malaria in 2000. The purpose of this study was to monitor in vivo efficacy of the current standard combination therapy against Plasmodium falciparum and P. vivax malaria. Studies were conducted between 2003 and 2005 in the Simbu, East Sepik, and Madang Provinces in Papua New Guinea according to the revised protocol of the World Health Organization (WHO) for assessment of antimalarial drug efficacy. Children between six months and seven years of age with clinically overt and parasitologically confirmed P. falciparum or P. vivax malaria were treated according to the new policy guidelines (i.e., AQ plus SP given to patients weighing < 14 kg and CQ plus SP given to patients weighing < 14 kg). Children were monitored up to day 28 and classified according to clinical and parasitological outcome as adequate clinical and parasitological response (ACPR), early treatment failure (ETF), late clinical failure (LCF), or late parasitological failure (LPF). For P. falciparum malaria, polymerase chain reaction (PCR)-corrected treatment failure rates up to day 28 ranged between 10.3% and 28.8% for AQ plus SP and between 5.6% and 28.6% for CQ plus SP, depending on the region and the year of assessment. Overall treatment failure rate with AQ or CQ plus SP for P. vivax malaria was 12%. Our results suggest that the current first-line treatment in Papua New Guinea is not sufficiently effective. According to the new WHO guidelines for the treatment of malaria, a rate of parasitological resistance greater than 10% in the two dominant malaria species in the country justifies a change in treatment policy.     

In vitro susceptibility and genetic variations for chloroquine and mefloquine in Plasmodium falciparum isolates from Thai-Myanmar border

In vitro drug susceptibility to chloroquine (CQ) and mefloquine (MF) were assessed in 39 P. falciparum isolates from the Thai-Myanmar border area. To further characterize CQ- and MF-resistance profiles in this area, we also analyzed pfcrt K76T mutation that is critical for CQ resistance, and pfmdr1 polymorphism that has an association with MF resistance. Eighteen isolates were successfully examined by in vitro tests for CQ, and 17 of them had resistance to the drug. Geometric mean concentration of CQ that inhibited the growth of parasites at 50% (IC50) was 371 +/- 227 nM (105-971 nM). Sixteen isolates were successfully examined by in vitro tests for MF, and 8 of them were resistant to the drug. Geometric mean of IC50 for MF was 41 +/- 31 nM (4-125 nM). Genotypes of drug resistance, such as pfcrt and pfmdr1 mutations, were also analyzed. All the 39 isolates had the same haplotype (CVIET) for PfCRT at its 72-76th amino acids. A pfmdr1 Y86 mutation was found in 95% of isolates. A pfmdr1 D1042 mutation was also present in 7 isolates, while no pfmdr1 Y1246 mutation was observed. These results indicated a correlation between CQ resistance and the pfcrt T76 and pfmdr1 Y86 mutations.