Chloroquine-resistant Plasmodium falciparum isolates from the Sudan lack two mutations in the pfmdr1 gene thought to be associated with chloroquine resistance.

Isolates of Plasmodium falciparum from 3 areas of the Sudan were recovered from cryopreservation in London and their chloroquine sensitivity was determined in vitro. Chloroquine resistance was detected in 6/6 isolates from Khartoum, 1/4 from Sennar and 3/3 from Gadarif, indicating that resistance is spreading. All the isolates were sensitive to mefloquine. Studies using blood spots on glass fibre discs and the polymerase chain reaction did not detect two mutations in the pfmdr1 gene, thought to be correlated with chloroquine-resistance, in any of the isolates studied.     

Prevalence of mutations in the antifolates resistance-associated genes (dhfr and dhps) in Plasmodium vivax parasites from Eastern and Central Sudan

Plasmodium vivax is the most geographically widespread species, and its burden has been increasingly documented in Eastern and Central Sudan. P. vivax becomes the crucial challenge during elimination programs; thus an effective treatment is necessary to prevent the development and the spread of resistant parasites. Therefore, the main objective of the present study was to provide data on the prevalence of molecular markers in two genes (pvdhfr and pvdhps) associated with SP resistance after nine years of AS + SP deployment among P. vivax parasites from Eastern and Central Sudan using PCR-RFLP. During 2012–2013, a number of 66 blood spots were obtained on filter paper. The samples were collected before treatment from febrile patients who were microscopically positive for P. vivax, from three states in Eastern and Central Sudan (Gezira, Gedarif, and Kassala). Mutations were detected in three codons of pvdhfr (I13L, S58R, and S117N) and none in pvdhps. The majority of P. vivax parasites had double mutations (58R/117N, 58%) in dhfr gene, while all parasites were wild type in dhps gene. In addition, limited distinct haplotypes (n = 4) were detected. In conclusion, the prevalence of mutations associated with SP resistance is low in Eastern and Central Sudan. Such information is necessary for guiding malaria control measures in the frame of Roll Back Malaria strategies for the elimination of malaria in the world.     

Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86Y.

The choice of partner drug is critical for artemisinine-based combination therapy (ACT) to remain effective and amodiaquine (AQ) is one important candidate to evaluate. We treated 81 children <5 years with uncomplicated Plasmodium falciparum malaria with AQ alone and related the treatment outcome to the possible selection of pfcrt 76T, 152T, 163S, 326S, pfmdr1 86Y and pfmrp 191H, 437S in recurrent infections (recrudescenses and re-infections) and to the blood concentration of desethylamodiaquine (DEAQ). During 21 days follow-up 28 children had a recurrent infection (9 recrudescenses, 13 re-infections and 6 mixed). Neither genotyping of the polymorphisms before treatment nor DEAQ blood concentrations could predict treatment outcome. pfcrt 76T was however significantly selected for in recurrent infections (p=0.020). pfmdr1 86Y was also selected for, but only in recrudescent infections (p=0.048). The study showed high prevalence of AQ resistant parasites in vivo, which appeared to be associated to pfcrt 76T and pfmdr1 86Y.     

Therapeutic response of multidrug-resistant Plasmodium falciparum and P. vivax to chloroquine and sulfadoxine-pyrimethamine in southern Papua, Indonesia

To determine the level of antimalarial drug resistance in southern Papua, Indonesia, we assessed the therapeutic efficacy of chloroquine plus sulfadoxine-pyrimethamine (CQ+SP) for Plasmodium falciparum infections as well as CQ monotherapy for P. vivax infections. Patients with P. falciparum failing therapy were re-treated with unsupervised quinine+/-doxycycline therapy and those with P. vivax with either unsupervised quinine+/-doxycycline or amodiaquine. In total, 143 patients were enrolled in the study (103 treated with CQ+SP and 40 with CQ). Early treatment failures occurred in four patients (4%) with P. falciparum and six patients (15%) with P. vivax. The failure rate by Day 28 for P. vivax was 65% (95% CI 49-81). After PCR correction for re-infections, the Day 42 recrudescence rate for P. falciparum infections was 48% (95% CI 31-65). Re-treatment with unsupervised quinine+/-doxycycline resulted in further recurrence of malaria in 48% (95% CI 31-65) of P. falciparum infections and 70% (95% CI 37-100) of P. vivax infections. Eleven patients with recurrent P. vivax were re-treated with amodiaquine; there were no early or late treatment failures. In southern Papua, a high prevalence of drug resistance of P. falciparum and P. vivax exists both to first- and second-line therapies. Preliminary data indicate that amodiaquine retains superior efficacy compared with CQ for CQ-resistant P. vivax.     

Application of a simplified in-vivo test system for determining chloroquine resistance in Plasmodium falciparum

A simplified in-vivo test system was applied to detect chloroquine resistance in malaria patients in Shahjahanpur district (Uttar Pradesh) in India. In 27.6% of cases RIII resistance was observed. This in-vivo method is a simple and useful test for the early detection of chloroquine-resistant falciparum infections and for the management of these patients with alternative therapy.     

DNA sequence polymorphisms of the pfmdr1 gene and association of mutations with the pfcrt gene in Indian Plasmodium falciparum isolates

Mutations in the Plasmodium falciparum multidrug resistance (pfmdr1) gene are known to provide compensatory fitness benefits to the chloroquine (CQ)-resistant malaria parasites and are often associated with specific mutations in the P. falciparum CQ resistant transporter (pfcrt) gene. Prevalence of the specific mutations in these two genes across different malaria endemic regions was mostly studies. However, reports on mutations in the pfmdr1 gene and their genetic associations with mutations in the pfcrt gene in Indian P. falciparum field isolates are scarce. We have sequenced a 560 bp region of pfmdr1 coding sequence in 64 P. falciparum isolates collected from different malaria endemic populations in India. Twenty out of these 64 isolates were laboratory cultured with known in vitro CQ sensitiveness (10 sensitive and 10 resistant). Three low frequency mutations (two non-synonymous and one synonymous) in the pfmdr1 gene were segregating in Indian isolates in addition to the predominant Y₈₆ and Y₁₈₄ ones, with high haplotype and nucleotide diversity in the field isolates in comparison to the cultured ones. No statistically significant genetic association between the mutations in the pfmdr1 and pfcrt gene could be detected; almost all observed associations were intragenic in nature. The results on the genetic diversity of the pfmdr1 gene were discussed in term of evolutionary perspectives in Indian P. falciparum, with possible future potential of gaining further insights on this gene in view of evolving malaria parasites resistant to artemisinin partner drugs.     

Mutations in Pfcrt and Pfmdr1 genes of Plasmodium falciparum isolates from two sites in Northcentral and Southwest Nigeria

The ability of malaria parasites to develop resistance to antimalarial drugs has made it necessary to continuously survey malaria parasite populations for resistance markers. Mutations in specific malaria parasite genes confer resistance to antimalarial drugs. The study compared mutations in Pfcrt and Pfmdr1 genes of P. falciparum from two ecologically different areas of Nigeria. Plasmodium falciparum dried blood spots collected from New Bussa (Northcentral Nigeria) and Ijede (Southwest Nigeria) were analysed by PCR-RFLP for Pfcrt, K76 T, Pfmdr1, N86Y and Y184F mutations. Pfmdr1 copy number was determined by quantitative-PCR. A total of 145 blood spots [Ijede = 55; New Bussa = 90 blood spots] were analysed, but Pfcrt gene was successfully amplified in 144 samples while Pfmdr1 was amplified in 132 samples. Overall, prevalence of mutant forms of Pfcrt 76 T, Pfmdr1 86Y and 184F were 74.3% (95% CI: 66.4–81.2%), 18.2% (95% CI: 12.0–25.8%) and 35.6% (95% CI: 27.5–44.4%). The frequency of Pfcrt 76 T was similar in both study sites [Ijede: 81.8% (95%CI: 69.1–90.9%); New Bussa: 69.7% (95%CI: 59.0–79.0), p = 0.105]. However, the frequencies of Pfmdr1 86Y and 184F were significantly higher in Ijede (28.3% and 62.3%) than in New Bussa (11.4% and 17.7%), respectively (P < 0.05). Eight parasite genotypes based on three codons of the two genes were identified. The most frequent genotype was TNY 53(40.5%) while the least was KYF 1 (0.8%). The most frequent genotype in Ijede and New Bussa were TNF 18(34.0%) and TNY 40 (51.3%) respectively. The frequency of wild strain KNF in Ijede and New Bussa were 3 (5.7%) and 18 (23.1%), respectively. The distribution of the genotypes differed significantly by location. The genotypes with more than two or more mutations were more in Ijede 32 (60.4%) than in New Bussa 16 (20.5%) (p < 0.001). Amplification of Pfmdr1 copy number was not observed in the two study sites. The prevalence of Pfcrt 76 T was similar in both locations while Pfmdr1 86Y and 184F differed in both locations. Single nucleotide polymorphisms in the three codons assessed were more in Ijede than in New Bussa.     

Chloroquine resistant Plasmodium falciparum on the island of Flores, Indonesia

The in vitro sensitivity of Plasmodium falciparum to chloroquine was studied in parasites from 45 children on the island of Flores, Indonesia. The micro in vitro culture technique and a 12-volt battery-operated field incubator were found to be well suited to the field situation encountered. Parasites from seven children (15.6%) were resistant to chloroquine in vitro: two at 8 pico-moles of chloroquine, two at 16 pico-moles, and three at 32 pico-moles. This is the first report of chloroquine resistant from Flores.     

Mutation analysis in pfmdr1 and pfmrp1 as potential candidate genes for artemisinin resistance in Plasmodium falciparum clinical isolates 4 years after implementation of artemisinin combination therapy in Iran

The emergence and spread of Plasmodium falciparum resistant to the commonly used anti-malarial drugs is a major challenge in the control and elimination of malaria. The present study provides information on genetic analysis in multidrug resistance 1 (pfmdr1) (N86Y/Y184F/S1034C/N1042D/F1226Y/D1246Y) and multidrug resistance protein 1 (pfmrp1) (H191Y/S437A/I876V/F1390I/K1466R) genes that are probably associated with artemisinin as well as chloroquine resistance transporter (pfcrt) 76T in P. falciparum clinical isolates (N = 200) exposed to artemisinin-based combination therapy (ACT) 4 years after its adoption in Iran. Also, the copy number of pfmdr1 gene was screened for its association with pfmdr1 mutations to incriminate artemisinin resistance. By using nested PCR-RFLP and sequencing analysis, none of the samples had any mutation at codons 1034, 1042, 1226 and 1246 of pfmdr1, while 86Y and 184F mutations were detected in 46% and 2% of the examined samples, respectively. Also, no significant difference was identified among analyzed samples collected before (baseline, 2002–2005) and after adoption of ACT (2007–2010) (P > 0.05). As with pfmrp1 gene, the mutations at positions 191Y (76.5%), 437A (69.5%), 876V (64.5%) and 1390I (17%) were detected and no samples displayed mutation at codon 1466R. In total, 42.5% of the examined isolates carried both pfmdr1 86Y and pfcrt 76T and none of the parasites simultaneously harbored pfcrt 76T, pfmdr1 86Y, 184F and pfmrp1 191Y, 437A, 876V, 1390I mutations. In addition, the copy number of pfmdr1 gene (N = 1) was similar as a sensitive isolate, 3D7, to artemisinin. In summary, none of the potential mutations associated with artemisinin and its derivatives resistance was significantly changed 4 years after adoption of ACT in Iran.     

Sequence Diversity of pfmdr1 and Sequence Conserve of pldh in Plasmodium falciparum from Indonesia: Its implications on Designing a Novel Antimalarial Drug with Less Prone to Resistance

Background

pfmdr1 and its variants are molecular marker which are responsible for antibiotics resistance in Plasmodium falciparum, a parasitic carrier for malaria disease. A novel strategy to treat malaria disease is by disrupting parasite lactate dehydrogenase (pLDH), a crucial enzyme for Plasmodium survival during their erythrocytic stages. This research was aimed to investigate and characterize the pfmdr1 and pldh genes of P. falciparum isolated from Nusa Tenggara Indonesia.

Methods

Genomic DNA of P.falciparum was isolated from malaria patients in Nusa Tenggara Indonesia. pfmdr1 was amplified using nested PCR and genotyped using Restriction Fragment Length Polymorphism (RFLP). pldh was amplified, sequenced, and analyzed using NCBI public domain databases and alignment using Clustal W ver. 1.83.

Results

Genotyping of the pfmdr1 revealed that sequence diversity was extremely high among isolates. However, a sequence analysis of pldh indicated that open reading frame of 316 amino acids of the gene showing 100% homology to the P. falciparum 3D7 reference pldh (GeneBank: {"type":"entrez-nucleotide","attrs":{"text":"XM_001349953.1","term_id":"124513265","term_text":"XM_001349953.1"}}XM_001349953.1).

Conclusion

This is the first report which confirms the heterologous of pfmdr1 and the homologous sequences of P.falciparum pldh isolated from Nusa Tenggara Islands of Indonesia, indicating that the chloroquine could not be used effectively as antimalarial target in the region and the pLDH-targeted antimalarial compound would have higher chance to be successful than using chloroquine for curbing malaria worldwide.     

Reversal of chloroquine resistance with desipramine in isolates of Plasmodium falciparum from Central and West Africa

Twenty freshly isolated strains of Plasmodium falciparum from non-immune and semi-immune patients infected in Central and West Africa were tested for in vitro drug sensitivity to chloroquine alone and to a combination of chloroquine and desipramine without prior adaptation to continuous culture. Combination of desipramine at 625 nmol/litre and chloroquine reversed resistance totally (IC50 less than 100 nmol/litre) in all 14 chloroquine-resistant isolates. No effect on chloroquine sensitivity was noted in the 6 chloroquine-sensitive isolates. These observations provide additional evidence that reversal of chloroquine resistance in the presence of desipramine is not a phenomenon unique to culture-adapted strains and that desipramine may exert the same potentiating effect in vivo, provided that effective plasma concentration can be attained without serious side-effects.     

Chloroquine resistance of Plasmodium falciparum is associated with severity of disease in Nigerian children

Chloroquine resistance of Plasmodium falciparum in vitro was significantly higher in isolates from patients with severe malaria than those with uncomplicated disease. This association may be due to either progression of uncomplicated to severe disease following chloroquine failure or increased virulence of chloroquine-resistant parasites. The implication of this for antimalarial treatment policy is discussed.     

Polymorphisms in Pfcrt and Pfmdr-1 genes after five years withdrawal of chloroquine for the treatment of Plasmodium falciparum malaria in West Bengal,India

BackgroundThe emergence of resistant power against different antimalarial agents particularly by Plasmodium falciparum is a challenge to combat malaria. Regular monitoring is essential not only to determine the efficacy and development of resistance by the parasite but also to detect early sign of regaining sensitivity to any anti-malarial agent that has been withdrawn for a long period. Studies on molecular markers associated with antimalarial drug resistance of prevailing Plasmodium population play an important role in this aspect. The present protocol was designed to study the polymorphisms in pfcrt and pfmdr-1 gene to determine any sign of regaining sensitivity to chloroquine among P. falciparum after five years of artemisinin combination therapy (ACT) implementation.MethodsClinical isolates were collected from P. falciparum positive patients attending the malaria clinic of Calcutta School of Tropical Medicine during December 2014 to December 2015. Genomic parasitic DNA was extracted and subjected to sequencing of pfcrt and pfmdr-1 gene directly from purified PCR products.ResultsA total of 89 isolates were sequenced for pfcrt and 73 isolates for pfmdr-1 genes. In pfcrt gene mutant K76T was detected in all isolates and all were SVMNT haplotype. Out of three important polymorphisms in pfmdr-1 gene mutant Y184F was detected among all isolates. One synonymous G182G and one non-synonymous S232F/Y, mutation were detected in 99% isolates.ConclusionAll isolates carrying mutant K76T in pfcrt gene, considered as hall mark for CQ resistance, indicate that there is no sign of regaining CQ sensitivity among the prevailing P. falciparum population of the study area after five years of ACT implementation.     

Field applications of agglutination and cytoadherence assays with Plasmodium falciparum from Papua New Guinea

Plasmodium falciparum isolates obtained directly from patients in Papua New Guinea were tested in their first cycle of growth in vitro for adherence to melanoma cells and for susceptibility to agglutination by immune serum. Binding varied among isolates and, in many cases, increased with further rounds of replication under optimal culture conditions. Binding inhibition assays and agglutination assays demonstrated extreme heterogeneity of surface antigens; apparently none of the sera from adult patients recognized all of the variants presented.     

氟喹诺酮耐药肠球菌gyrA和parC基因突变特征及其与环丙沙星耐药相关性分析

目的 探讨氟喹诺酮耐药肠球菌的gyrA和parC基因突变特征与环丙沙星耐药程度的相关性。方法 收集2016 - 2018年临床分离59株粪肠球菌和62株屎肠球菌,使用PCR方法特异性扩增gyrA和parC基因,并进行基因测序检测分析。结果 粪肠球菌和屎肠球菌对环丙沙星耐药率分别为59.32%和80.65%。粪肠球菌gyrA基因Ser83突变（χ2 = 51.252, P＜0.001）和parC基因Ser80突变（χ2 = 55.115, P＜0.001）与粪肠球菌对环丙沙星高水平耐药（MIC≥16μg/ml）的相关性差异具有统计学意义;屎肠球菌gyrA基因Ser83突变（χ2 = 42.014, P＜0.01）和parC基因Ser80突变（χ2 = 62.000, P＜0.01）与屎肠球菌对环丙沙星高水平耐药（MIC≥16 μg/ml）的相关性差异具有统计学意义。结论 gyrA基因Ser83突变和parC基因Ser80突变与肠球菌对环丙沙星高水平耐药（MIC≥16 μg/ml）可能相关。