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.     

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 prophylaxis associated with high prevalence of Plasmodium falciparum pfcrt K76T mutation in people with sickle-cell disease in Benin City, Nigeria

BACKGROUND & OBJECTIVES: High mortality and morbidity in sickle-cell disease has been associated with malaria infection especially in countries where chloroquine is used. Chloroquine resistance has been associated with the emergence of Pfcrt mutant genes. This study aimed at comparing the prevalence rate of Pfcrt T76 mutation in Plasmodium falciparum isolates from infected individuals with sickle-cell disease and sickle-cell trait. This study was carried out in Benin City between the months of April and June 2006. This period is marked with high transmission rate of malaria. METHODS: The genotype of the subjects was screened using haemoglobin electrophoresis system and the P. falciparum. Pfcrt genotyping was carried out using PCR-restriction fragment length polymorphism (RFLP). RESULTS: Four hundred and twenty-four subjects comprising of 207 haemoglobin AA, 136 haemoglobin AS and 81 haemoglobin SS typed individuals were enrolled for this study. No significant difference existed in the prevalence rate of malaria in the three groups (p > 0.05). However, the prevalence rate of Pfcrt K76T mutant gene was higher in the haemoglobin SS genotyped individuals than the haemoglobin AA and AS subjects (p < 0.05). INTERPRETATION & CONCLUSION: An uncontrolled use of chloroquine has been incriminated as the major cause of chloroquine resistance in Nigeria. Therefore, rapid intervention measures are needed as a matter of urgency to curb the up rise in the prevalence of the chloroquine resistant genes in our environment.     

海南恶性疟原虫分离株Pfcrt和Pfmdr1基因点突变的研究

目的 分析海南省恶性疟原虫分离株氯喹抗性转运蛋白编码基因(Pfcrt)及其P-糖蛋白同系物1（Pgh1)编码基因(Pfmdr1)的点突变特征， 为探讨抗性分子标记用于氯喹抗性监测提供参考。 方法 采用巢式PCR（nested-PCR）和限制性酶切片段长度多态性（RFLP）方法， 检测Pfcrt基因编码第76位氨基酸的密码子和Pfmdr1基因编码第86、1246位氨基酸的密码子发生点突变情况。按世界卫生组织（WHO）推荐的体外微量法测定恶性疟原虫对氯喹的敏感性。 结果 检测的36份血样中， 28份成功扩增Pfcrt基因， 导致第76位氨基酸由赖氨酸（K）变为苏氨酸（T）的突变型占64.3%， 混合型占14.3%， 野生型占21.4%； 29份成功扩增Pfmdr1基因， 导致第86位氨基酸由天冬酰胺（N）变为酪氨酸（Y）的突变型占3.4%， 混合型占6.9%, 野生型占89.7%。未发现编码第1246位氨基酸的密码子发生点突变。体外氯喹敏感试验结果显示, 72.2%(26/36)分离株存在抗性。 治疗前恶性疟原虫Pfcrt 76T突变发生率在体外微量测定法显示的氯喹抗性与敏感株中的差异有统计学意义（P＜0.05）， 而Pfmdr1点突变发生率在氯喹抗性与敏感株中的差异无统计学意义（P＞0.05）。 结论 恶性疟原虫Pfcrt 基因76T可以作为监测氯喹抗性的一个分子标记。     

我国恶性疟原虫Pfcrt和Pfmdr1基因多态性及与氯喹敏感性关系的研究

目的了解我国恶性疟原虫分离株Pfcrt基因K76T及Pf mdr1基因N86Y和D1246Y的点突变特征及发生率,并分析上述分子标志与恶性疟原虫对氯喹敏感性的关系。方法从我国恶性疟流行区云南和海南省采集恶性疟现症患者血样,根据恶性疟原虫Pfcrt基因和Pf mdr1基因序列设计巢式PCR引物,以血样中的恶性疟原虫DNA为模板,进行巢式PCR-RFLP分析,并对部分PCR产物进行测序验证。采用世界卫生组织制定的体外微量法测定同一批血样中恶性疟原虫对氯喹的敏感性。结果云南、海南省恶性疟原虫Pfcrt基因K76T的突变发生率分别为86.7%和64.3%;云南、海南省恶性疟原虫Pf mdr1 N86Y突变发生率分别为46.5%和3.4%。未发现云南、海南省恶性疟原虫分离株存在Pf mdr1 D1246Y突变。体外微量测定法显示Pfcrt 76T突变发生率氯喹抗性株与敏感株间差异有统计学意义（χ^2=24.70,P〈0.01）。Pf mdr1 86Y突变发生率氯喹抗性株与敏感株间差异无统计学意义（χ^2=0.20,P=0.65）。结论在云南省和海南省现场未发现恶性疟原虫Pf mdr1 D1246Y点突变,抗氯喹恶性疟原虫的Pf mdr1 N86Y突变发生率与敏感株间无显著差异。Pfcrt K76T作为分子标志在我国恶性疟原虫氯喹抗性监测中具有应用价值。     

Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene haplotype SVMNT in P. falciparum malaria in India

The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) and the P. falciparum multidrug resistance 1 (Pfmdr1) mutation (N86Y) were analyzed as markers of chloroquine resistance in the DNAs of 73 blood samples from patients with P. falciparum malaria in India. Seventy of the 73 DNAs had the Pfcrt K76T mutation. Of these, 66 had the SVMNT haplotype and four had CVIET, the African/Southeast Asian haplotype. Only 20 of 69 DNAs had the Pfmdr1 N86Y mutation. It is surprising that the Pfcrt haplotype in India is predominantly SVMNT, rather than that seen in Southeast Asia. The widespread prevalence of the Pfcrt K76T mutation is a cause for concern.     

2012年和2018年我国输入性恶性疟原虫氯喹抗性基因多态性分析

目的对2012年和2018年我国输入性恶性疟原虫样本氯喹抗性分子标记位点基因多态性进行检测,分析恶性疟原虫氯喹抗性转运蛋白基因(Plasmodium falciparum chloroquine re sistant transporter,Pfcrt)第72~76位密码子抗性相关位点突变类型,并分析不同输入来源样本的特异性。方法收集2012年和2018年国家疟疾诊断参比实验室674例输入性恶性疟病例滤纸血样本,以样本中恶性疟原虫7号染色体上Pfcrt基因第72~76位点为扩增片段,采用巢式PCR法进行扩增并测序,对目的产物片段测序结果、地理分布等特征进行统计分析。结果2012年和2018年我国674例输入性恶性疟病例中,95.5%(644/674)来自非洲,其余4.5%(30/674)来自东南亚和大洋洲(巴布亚新几内亚);非洲又以西非和中非为主(占非洲样本的80.4%,518/644)。共检测到C72S、M74I、N75E、K76T 4个位点突变和5种单体型类型(CVMNK、CVIET、SVMNT和两种混合型),其中CVMNK与CVIET为非洲和东南亚地区恶性疟原虫共有的单体型类型,SVMNT仅在东南亚(缅甸)和巴布亚新几内亚输入样本中检测出;2种混合型为CVMNK/CVIET和CVMNK/SVMNT,前者在非洲和东南亚输入样本中分布,后者仅在东南亚缅甸来源样本中检出。自非洲输入的样本野生型较多,占77.7%(478/615);而自东南亚和巴布亚新几内亚输入的样本中,抗性分子标记样本占68.0%(17/25),两者差异有统计学意义(χ^2=28.5,P<0.05)。非洲不同地区来源样本中,抗性基因比例和野生型构成差异有统计学意义(P<0.01),西非野生型所占比例最低。结论2012年和2018年我国674例输入性恶性疟病例样本中,自东南亚输入的恶性疟原虫Pfcrt基因第72~76位点抗性基因比例和分子多态性均较非洲来源样本高。     

输入性恶性疟原虫Pfcrt基因76位点多态性分析

目的 目的 了解输入性恶性疟原虫氯喹抗性转运蛋白基因（Pfcrt基因）K76T点突变发生情况。方法 方法 采集 2008-2012年从非洲、 东南亚等疟疾流行区回国人员恶性疟现症患者血样, 根据恶性疟原虫Pfcrt基因序列设计巢式PCR引 物, 以血样中的恶性疟原虫DNA为模板, 进行巢式PCR?RFLP分析。结果 结果 92份输入性恶性疟现症患者血样中, 突变型Pf? crt基因50份, 占54.3%; 野生型Pfcrt基因42份, 占45.7%。采自非洲国家 （尼日利亚、 赤道几内亚、 利比里亚等9国） 回国人 员的70份血样中, 野生型Pfcrt基因37份, 突变型Pfcrt基因33份, 分别占52.9%和47.1%; 采自东南亚国家 （缅甸和印度尼西 亚） 回国人员的22份血样中, 野生型Pfcrt基因5份, 突变型Pfcrt基因17份, 分别占22.7%和77.3%。不同地区回国人员Pfcrt 基因突变发生率差异有统计学意义 （χ2 = 6.12, P < 0.05）。结论 结论 来自不同流行区的恶性疟原虫分离株Pfcrt基因突变发 生率也不同, Pfcrt基因K76T位点突变检测在输入性恶性疟原虫氯喹抗性监测中具有应用价值。     

中国恶性疟原虫氯喹抗药性相关基因Pfcrt 72-76位点基因型分析

目的了解我国云南和海南恶性疟原虫分离株Pfcrt基因72-76位点基因型,并分析该位置不同基因型与恶性疟原虫对氯喹敏感性的关系。方法从我国恶性疟流行区云南和海南省采集恶性疟现症患者血样,根据恶性疟原虫Pfcrt基因序列设计引物,进行巢式PCR扩增和测序。采用WHO制定的体外微量法测定同批血样中恶性疟原虫对氯喹的敏感性。结果云南、海南省恶性疟原虫Pfcrt基因第72-76位编码的氨基酸单元型在氯喹敏感株均为CVMNK。其中海南的氯喹抗性株Pfcrt基因第72-76位编码的氨基酸为单一的CVIET单元型,云南氯喹抗性株中除一株为CVIKT,另1株为SVMNT外,均为CVIET单元型。结论我国云南、海南省恶性疟原虫氯喹敏感株Pfcrt基因第72-76位编码的氨基酸基因型为CVMNK。抗性株主要为CVIET型,Pfcrt基因第72-76位编码氨基酸基因型可作为氯喹抗性分子标志。     

Usefulness of the Plasmodium falciparum chloroquine resistance transporter T76 genotype failure index for the estimation of in vivo chloroquine resistance in Burkina Faso

The prevalence of chloroquine (CQ) treatment failure and the genotype failure index was determined in four sentinel sites in Burkina Faso. In three sites, the genotype failure index varied between 1.7 and 3, a result confirming the relationship between the Plasmodium falciparum CQ resistance transporter (Pfcrt) T76 mutation and CQ resistance. In the remaining site, the genotype failure index was unusually low, 1.1, which was significantly different than that in the other sites (P < 0.00001). These findings are discussed. Often but not always, the prevalence of CQ resistance can be correctly estimated by the Pfcrt T76 genotype failure index.     

Monitoring chloroquine resistance using Plasmodium falciparum parasites isolated from wild mosquitoes in Tanzania

Monitoring antimalarial drug resistance is a useful epidemiologic tool and provides early detection of resistance foci. Using DNA extracted from the head/thorax of wild mosquitoes collected from Bagamoyo Coastal Tanzania, samples infected by Plasmodium falciparum (N = 89, in 2002 and N = 249 in 2004) were screened by nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for mutations at Pfcrt76 and Pfmdr1-86 associated with chloroquine (CQ) resistance. The majority of isolates were of single infection (71%), and the prevalence of mutant alleles of Pfcrt76 decreased from 64.5% in 2002 to 16% in 2004; likewise, mutant Pfmdr1-86 alleles decreased from 46.6% to 2.7%. Overall, there was a decline of mutant isolates by a factor of 17 and 4 for Pfmdr1 and Pfcrt, respectively. In contrast, isolates with wild-type alleles increased significantly from < 20% in 2002 to 67.6% for Pfcrt76 and 83.5% for Pfmdr1-86 in 2004. This observation suggest a biologic trend of decrease of CQ mutants and a subsequent increase of CQ susceptible parasites in circulation after the discontinued use of CQ in 2001 as a first-line drug in Tanzania. High prevalence of susceptible P. falciparum found in circulation not only supports other reports of a decline of mutant parasites after a reduction of drug selection pressure but suggests that the fitness cost is high in mutant parasites. Typing parasite isolates from infected mosquitoes, an alternative means of data collection, has the potential to increase the spatial and temporal coverage, and this approach is practical in highly endemic regions of Africa.     

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.     

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.     

Changes in genotypes of Plasmodium falciparum human malaria parasite following withdrawal of chloroquine in Tiwi, Kenya

Chloroquine (CQ) drug was withdrawn in 1998 as a first-line treatment of uncomplicated malaria in Kenya. This was in response to resistance to the drug in Plasmodium falciparum malaria parasite. Investigations were conducted to determine prevalence of CQ resistance genotypes in the parasites in Tiwi, a malaria endemic town in Kenya, before and about a decade after the withdrawal of the drug. Blood samples were collected and spotted on filter papers in 1999 and 2008 from 75 and 77 out-patients respectively with uncomplicated malaria. The sampling was conducted using finger pricking technique. DNA was extracted from individual spots in the papers and screened for the presence of P. falciparum chloroquine resistance transporter (Pfcrt) and multi drug resistance (Pfmdr-1) markers using nested PCR. Nature of mutations (haplotypes) in the Pfcrt and Pfmdr-1 markers in the samples were confirmed using dot blot hybridization technique. Changes in pattern of CQ resistance in the parasite samples in 1999 and 2008 were assessed by Chi Square test. There was a significant (P<0.05) reduction in CQ resistant genotypes of the parasite between 1999 and 2008. Pfmdr and Pfcrt CQ resistant genotypes in 2008 reduced to 54.10 and 63.64% respectively, from 75.39 and 88.0% respectively in 1999. This reduction was accompanied by emergence of Pfcrt specific CQ sensitive (IEK) and intermediate/partially CQ resistant (MET) haplotypes. Results suggest significant reversal of the phenotype of the parasite from chloroquine resistant to wild/sensitive type. The novel haplotypes indicates transitional phase of the parasite to the wild type. Current prevalence of chloroquine resistant genotype is definitely above the threshold for efficacious re-introduction of chloroquine for treatment of malaria in Tiwi.     

Antimalarial drug sensitivity profile of western Kenya Plasmodium falciparum field isolates determined by a SYBR Green I in vitro assay and molecular analysis

In vitro drug sensitivity and molecular analyses of Plasmodium falciparum track drug resistance. DNA-binding fluorescent dyes like SYBR Green I may allow field laboratories, proximal to P. falciparum collection sites, to conduct drug assays. In 2007-2008, we assayed 121 P. falciparum field isolates from western Kenya for 50% inhibitory concentrations (IC(50)) against 6 antimalarial drugs using a SYBR Green I in vitro assay: 91 immediate ex vivo (IEV) and 30 culture-adapted, along with P. falciparum reference clones D6 (chloroquine [CQ] sensitive) and W2 (CQ resistant). We also assessed P. falciparum mdr1 (Pfmdr1) copy number and single nucleotide polymorphisms (SNPs) at four codons. The IC(50)s for IEV and culture-adapted P. falciparum isolates were similar, and approximated historical IC(50)s. For Pfmdr1, mean copy number was 1, with SNPs common at codons 86 and 184. The SYBR Green I assay adapted well to our field-based laboratory, for both IEV and culture-adapted P. falciparum, warranting continued use.     

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.     

Madagascan isolates of Plasmodium falciparum showing low sensitivity to artemether in vitro

In Madagascar, although chloroquine (CQ) remains the first-line treatment of choice for malaria, the gradual spread of resistance to this antimalarial drug is of increasing concern. As part of a larger investigation of the effectiveness of the second- and third-line drugs used to treat malaria, the in-vitro susceptibilities of Plasmodium falciparum collected in Madagascar to CQ, mefloquine (MQ) and artemether (ART) were therefore investigated. Median inhibitory concentrations (IC(50)) were determined for isolates collected from residents of two villages in the foothills of the central highlands. The IC(50) for ART ranged from 0.23-17.50 nM [N = 51; geometric mean = 4.02 nM; 95% confidence interval (CI) = 2.99-5.05 nM], four isolates exhibiting IC(50) (> 12 nM) indicative of resistance to this drug. The artemether IC(50) were found to be correlated with those of CQ (N = 46; Spearman's r = 0.51; P = 0.0002), which varied widely (0.4-254.3 nM; mean = 23.4 nM; CI = 7.1-39.7 nM; N = 46). Five (11%) of the 46 isolates exposed to CQ in vitro were considered resistant to this drug (i.e. to have IC(50) > 100 nM), with IC(50) ranging from 109-245.3 nM (mean = 171.6 nM; CI = 110.4-232.8 nM). However, all the CQ-resistant isolates were considered sensitive to ART and vice versa. All the isolates tested also appeared sensitive to MQ (IC(50) = 2.21-43.1 nM; mean = 10.5 nM; CI = 7.95-13.07 nM; N = 46), the IC(50) for MQ being correlated with those for CQ (N = 46; Spearman's r =0.46; P = 0.001). There was no significant correlation between ART and MQ activities. Although the sample was fairly small, the present results indicate that P. falciparum in Madagascar is generally becoming less sensitive to CQ and ART. The observation of a correlation between the IC(50) for these two drugs perhaps indicates that artemisinin derivatives would be better used in combination with antimalarial drugs other than 4-aminoquinolines.     

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.     

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.     

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.