Clearance of drug-resistant parasites as a model for protective immunity in Plasmodium falciparum malaria

Residents of malaria-endemic areas sometimes spontaneously clear Plasmodium falciparum infection without drug treatment, implying an important role for host factors such as immunity in this clearance. Host factors may also contribute to clearance of parasites resistant to a treatment drug. Chloroquine resistance is caused by point mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene. We investigated the clearance of malaria parasites carrying the key chloroquine resistance-conferring PfCRT mutation K76T in patients treated with chloroquine. We found that the ability to clear these resistant parasites is strongly dependent on age (the best surrogate for protective immunity in endemic areas), suggesting that host immunity plays a critical role in the clearance of resistant P. falciparum infections. Age-adjusted comparison of subjects able to clear resistant parasites and those unable to do so provides a new phenotype for identifying host immune and genetic factors responsible for protective immunity against malaria.     

Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People's Republic of China

The use of chloroquine treatment for Plasmodium falciparum malaria was abandoned in China in 1979 because of widespread drug resistance. Subsequent studies found decreases in the prevalence of chloroquine-resistant strains. To evaluate these decreases and assess the current status of chloroquine sensitivity in Hainan, China, we determined the prevalence of the P. falciparum chloroquine resistance transporter (PfCRT) 76T marker in the DNA of blood samples collected from 1978 to 2001. Results showed the presence of PfCRT 76T in 101 of 112 samples (90%) from 1978 to 1981, 30 of 43 samples (70%) from 1986, 22 of 34 samples (65%) from 1997 to 1998, and 37 of 68 samples (54%) from 2001. The prevalence of PfCRT 76T thus progressively decreased after chloroquine was discontinued as a treatment for P. falciparum malaria (chi(2) = 5.2, P < 0.022 [1978-1981 versus 1986]; chi(2) = 7.4, P < 0.006 [1978-1981 versus 1997-1998]; and chi(2) = 28.8, P < 0.0001 [1978-1981 versus 2001]). Reduced prevalence of the PfCRT 76T marker is consistent with greater rates of chloroquine sensitivity from in vitro drug assays of blood samples in 1997 and 2001. Monitoring for continued decreases will provide valuable information for future drug-use policies in China.     

2018—2019年赤道几内亚Bioko岛恶性疟原虫抗药性基因多态性分析

目的　调查赤道几内亚Bioko岛恶性疟原虫多药耐药蛋白1（Plasmodium falciparum multidrug resistance protein 1, PfMDR1）、氯喹抗性转运蛋白基因（P. falciparum chloroquine resistant transporter, PfCRT）和Kelch 13基因（P. falciparum Kelch 13, PfK13）多态性,为当地疟疾防控策略制定提供参考。方法　采集2018—2019年赤道几内亚Bioko岛恶性疟原虫感染者外周血样本85份,提取基因组DNA。采用巢式PCR技术扩增PfMDR1、PfCRT和 PfK13基因,对扩增产物进行DNA测序,并对基因序列进行比对。结果　赤道几内亚Bioko岛恶性疟原虫PfK13基因不存在已知与青蒿素抗性相关的突变;PfMDR1基因和PfCRT基因均存在不同比例抗药性突变,其中PfMDR1_N86Y、PfMDR1_Y184F和PfCRT_K76T突变率分别为35.29%（30/85）、72.94%（62/85）和24.71%（21/85）。结论　赤道几内亚Bioko岛恶性疟原虫PfMDR1、PfCRT基因和 PfK13基因均存在不同程度突变。     

Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border

BACKGROUND: The combination of atovaquone and proguanil (Malarone) has been established as a drug of choice to prevent and treat multi-drug resistant Plasmodium (P.) falciparum malaria in travelers. However, several cases of resistance against Malarone have been reported in some parts of Africa, and many of the cases are believed to be associated with mutations at the codon 268 of cytochrome b gene in mitochondria of P. falciparum. The aim of the study was to estimate the effectiveness of Malarone in treatment and prophylaxis for the travelers to Thai-Myanmar border where multi-drug resistant malaria is highly endemic. METHODS: Seventy P. falciparum samples obtained from patients from Thai-Myanmar border were sequenced to detect mutations around the codon 268. The same samples were also sequenced to detect P. falciparum chloroquine resistance transporter mutation (PfCRT K76T). RESULTS: All the 70 samples showed no mutations at the codon 268 of cytochrome b gene. Whereas, 50 samples, whose pfcrt genes were sequenced successfully, had an identical genotype for K76T mutation. CONCLUSION: In Asian countries, even in the multi-drug resistant areas in the great Mekong region, no case of Malarone resistance has been reported clinically or genetically thus far. In this study, all the P. falciparum parasites tested successfully were shown to be chloroquine resistant but atovaquone susceptible genetically. The more the usefulness of Malarone increases for both treatment and prophylaxis, the wider the drug-resistance against Malarone may spread in the region. Although the total number of samples examined is not large, it is concluded from these findings that Malarone should be recommended for prophylaxis of malaria for travelers to the Mekong region.     

Evidence that mutant PfCRT facilitates the transmission to mosquitoes of chloroquine-treated Plasmodium gametocytes

Resistance of the human malarial parasite Plasmodium falciparum to the antimalarial drug chloroquine has rapidly spread from several independent origins and is now widely prevalent throughout the majority of malaria-endemic areas. Field studies have suggested that chloroquine-resistant strains might be more infective to mosquito vectors. To test the hypothesis that the primary chloroquine resistance determinant, mutations in PfCRT, facilitates parasite transmission under drug pressure, we have introduced a mutant or wild-type pfcrt allele into the rodent model malarial parasite Plasmodium berghei. Our results show that mutant PfCRT from the chloroquine-resistant 7G8 strain has no effect on asexual blood stage chloroquine susceptibility in vivo or ex vivo but confers a significant selective advantage in competitive mosquito infections in the presence of this drug, by protecting immature gametocytes from its lethal action. Enhanced infectivity to mosquitoes may have been a key factor driving the worldwide spread of mutant pfcrt.     

Polymorphisms in the Plasmodium falciparum pfcrt and pfmdr-1 genes and clinical response to chloroquine in Kampala, Uganda

The molecular mechanism of chloroquine resistance in Plasmodium falciparum remains uncertain. Polymorphisms in the pfcrt and pfmdr-1 genes have been associated with chloroquine resistance in vitro, although field studies are limited. In evaluations of known polymorphisms in parasites from patients with uncomplicated malaria in Kampala, Uganda, the presence of 8 pfcrt mutations and 2 pfmdr-1 mutations did not correlate with clinical response to therapy with chloroquine. Most notably, the pfcrt lysine-->threonine mutation at position 76, which recently correlated fully with chloroquine resistance in vitro, was present in 100% of 114 isolates, of which about half were from patients who recovered clinically after chloroquine therapy. These results suggest that, although key pfcrt polymorphisms may be necessary for the elaboration of resistance to chloroquine in areas with high levels of chloroquine resistance, other factors, such as host immunity, may contribute to clinical outcomes.     

Origin and dissemination of Plasmodium falciparum drug-resistance mutations in South America

Multidrug resistance is a major obstacle to the control of Plasmodium falciparum malaria, and its origins and modes of dissemination are imperfectly understood. In this study, haplotyping and microsatellite analysis of malaria from 5 regions of the South American Amazon support the conclusion that the parasite mutations conferring mid- and high-level resistance to the antifolate combination sulfadoxine-pyrimethamine have a common origin. Parasites harboring these mutations are also found to share drug-resistance alleles that confer a unique chloroquine resistance phenotype and to be similar at loci not linked to drug resistance, although not genetically identical. Since the 1980s, multidrug-resistant P. falciparum has spread in a north-northwest manner across the continent, from an origin likely in the lower Amazon. This study highlights the importance of continent-wide malaria-control policies and suggests that the containment of resistance to the next generation of therapies may be feasible.     

Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi

In 1993, Malawi stopped treating patients with chloroquine for Plasmodium falciparum malaria because of a high treatment failure rate (58%). In 1998, the in vitro resistance rate to chloroquine was 3% in the Salima District of Malawi; in 2000, the in vivo resistance rate was 9%. We assayed two genetic mutations implicated in chloroquine resistance (N86Y in the P. falciparum multiple drug resistance gene 1 and K76T in the P. falciparum chloroquine resistance transporter gene) in 82 P. falciparum isolates collected during studies in 1998 and 2000. The prevalence of N86Y remained similar to that in neighboring African countries that continued to use chloroquine. In contrast, the prevalence of K76T was substantially lower than in neighboring countries, decreasing significantly from 17% in 1998 to 2% in 2000 (P < 0.02). However, neither mutation was significantly associated with in vivo or in vitro resistance (P > 0.29). Withdrawal of the use of chloroquine appears to have resulted in the recovery of chloroquine efficacy and a reduction in the prevalence of K76T. However, other polymorphisms are also expected to contribute to resistance.     

Molecular diagnosis of resistance to antimalarial drugs during epidemics and in war zones

Plasmodium falciparum mutations pfcrt K76T and the dhfr/dhps "quintuple mutant" are molecular markers of resistance to chloroquine and sulfadoxine-pyrimethamine, respectively. During an epidemic of P. falciparum malaria in an area of political unrest in northern Mali, where standard efficacy studies have been impossible, we measured the prevalence of these markers in a cross-sectional survey. In 80% of cases of infection, pfcrt K76T was detected, but none of the cases carried the dhfr/dhps quintuple mutant. On the basis of these results, chloroquine was replaced by sulfadoxine-pyrimethamine in control efforts. This example illustrates how molecular markers for drug resistance can provide timely data that inform malaria-control policy during epidemics and other emergency situations.     

The resistance of Plasmodium falciparum strains to preparations of the 4-aminoquinoline series in the Republic of Guinea]

In vivo measurements of P. falciparum sensitivity to chloroquine diphosphate, carried out in the Republic of Guinea, resulted in isolation of strains with degrees I and II resistance from 5 of the 25 examined patients, that may be due to a wide prophylactic administration of the drug to all patients with fevers without completing the course of therapy in all the patients infected. Such courses are obligatory to prevent the dissemination of chloroquine-resistant strains of the parasite and augmentation of the resistance. Further use of chloroquine and its analogs for the prevention and chemoprophylaxis of malaria in the Republic of Guinea appears still desirable, because the majority of P. falciparum strains are still sensitive to these agents. If no favorable changes are detectable in the clinical parameters of malaria patients in 4-5 days of chloroquine treatment or the condition recurs in 1.5-2 months, assessment of P. falciparum sensitivity to chloroquine is advisable, followed by a rational choice of the drug.     

Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria

This study investigated the association between Plasmodium falciparum chloroquine resistance transporter (pfcrt) T76 and P. falciparum multidrug resistance gene 1 (pfmdr1) Y86 alleles and in vivo amodiaquine (AQ) resistance, as well as the clearance of parasites harboring these two alleles in children treated with AQ in southwest Nigeria. One hundred one children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of AQ and followed-up for 28 days. Blood samples were collected on filter paper samples at enrollment and during follow-up for identification of parasite genotypes and pfcrt and pfmdr1 mutations using polymerase chain reaction and restriction fragment length polymorphism approaches. Parasitologic assessment of response to treatment showed that 87% and 13% (RI) of patients were cured and failed treatment, respectively. Although infections in patients were polyclonal (as determined by merozoite surface protein 2 genotyping), the presence of both mutants pfcrtT76 and pfmdr1Y86 alleles in parasites is associated with in vivo AQ resistance (odds ratio = 7.58, 95% confidence interval = 1.58-36.25, P = 0.006) and is selected by the drug in children who failed AQ treatment. Treatment failure with the combination of mutant pfcrtT76 and pfmdr1Y86 alleles as well as the ability of patients to clear these resistant parasites is dependent on age, suggesting a critical role of host immunity in clearing AQ-resistant P. falciparum. The combination of mutant pfcrtT76 and pfmdr1Y86 alleles may be useful markers for monitoring the development and spread of AQ resistance, when combining this drug with other antimalarials for treatment of malaria in Africa.     

Avian mortality surveillance for West Nile virus in Colorado

A total of 248 Plasmodium falciparum isolates were sampled in travelers with malaria who came to Marseille, France from Comoros to investigate in vitro activities of antimalarial drugs and molecular markers of drug resistance. Of the 248 isolates, 126 were maintained in culture. Of these, 53% were resistant to chloroquine, and 3% had reduced susceptibility to quinine, mefloquine, and atovaquone; 1% had reduced susceptibility to halofantrine and dihydroartemisinin; 7% had reduced susceptibility to monodesethylamodiaquine; 37% had reduced susceptibility to cycloguanil; and none had reduced susceptibility to lumefantrine. Resistance-associated point mutations were screened in 207 isolates. No mutations in the cytochrome b gene were found. Of the 207 isolates, 119 (58%) had a mutation in the P. falciparum dihydrofolate reductase (Pfdhfr) gene at codon 108, 6 (5%) had mutations in both Pfdhfr codon 108 and the P. falciparum dihydropteroate synthase codon 437, and 115 (56%) had the chloroquine resistance-associated K76T mutation in the P. falciparum chloroquine resistance transporter gene. This study represents a unique opportunity to improve surveillance of P. falciparum drug resistance in Comoros with consequences for treatment and chemoprophylaxis guidelines.     

Efficacy and safety of CGP 56697 (artemether and benflumetol) compared with chloroquine to treat acute falciparum malaria in Tanzanian children aged 1–5 years

A randomized, open trial involving 260 Tanzanian children, aged 1–5 years, with acute Plasmodium falciparum malaria was conducted to evaluate the efficacy of the combination antimalarial CGP 56697 (artemether and benflumetol), and to compare it with chloroquine, the standard drug used for malaria treatment in the Kilombero area. Children who had received rescue medication within the first 48 h or had a negative slide at the same time were excluded. Seven-day parasitological cure rates were 94% (95% CI 88–97.5) for CGP 56697 and 35.4% (95% CI 25.9–45.8) for chloroquine. Using the same definition, the 14-day parasitological cure rates were 86.4% (95% CI 78.5–92.2) for CGP 56697 and 10.3% (95% CI 5.1–18.1) for chloroquine. Gametocytes were more effectively suppressed by CGP 56697 than by chloroquine. There were no major adverse events with either drug. CGP 56697 is highly efficacious against P. falciparum in this area of Tanzania. The study contributes to the discussion on treatment strategies, particularly whether chloroquine may still fulfil its role as first-line drug in an area of high malaria transmission and very high levels of chloroquine resistance.     

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.     

The PfCRT (K76T) point mutation favours clone multiplicity and disease severity in Plasmodium falciparum infection

In Orissa, a malaria-hyperendemic area of India, we assessed the relationship between the PfCRT (K76T) point mutation of Plasmodium falciparum and the clinical severity of malaria. Forty uncomplicated and 36 severe malaria cases were selected, and parasite species, density and schizontaemia determined by examination of Giemsa-stained thick or thin blood films. The PfCRT point mutation was analysed by PCR-RFLP and genotypes of the parasite isolates investigated by nested PCR using the polymorphic region of the merozoite surface protein-2. We found that (i) the prevalence of the PfCRT point mutation was significantly higher (P < 0.01) in severe malaria cases and that (ii) heavy parasitaemia along with clone multiplicity was statistically more common (P < 0.01) in severe cases. These associations may be due to progression of uncomplicated to severe disease after chloroquine treatment failure and/or increased virulence of chloroquine-resistant parasites. The implications for antimalarial treatment policy are discussed.     

Chloroquine/doxycycline combination versus chloroquine alone, and doxycycline alone for the treatment of Plasmodium falciparum and Plasmodium vivax malaria in northeastern Irian Jaya, Indonesia.

Combination therapy is one method of overcoming the global challenge of drug-resistant Plasmodium falciparum malaria. We conducted a hospital-based 28-day in vivo test comparing chloroquine/doxycycline to chloroquine or doxycycline alone for treating P. falciparum and Plasmodium vivax malaria in Irian Jaya, Indonesia. Eighty-nine patients with uncomplicated falciparum malaria were randomized to standard dose chloroquine (n = 30), doxycycline (100 mg every 12 hours [7 days], n = 20), or chloroquine with doxycycline (n = 39); corresponding numbers for vivax malaria (n = 63) were 23, 16, 24. Endpoints were parasite sensitivity (S) or resistance (RI/RII/RIII). Of the 105 evaluable patients, chloroquine/doxycycline cured (S) 20/22 (90.9% [95% CI 78.9-100%]) patients with P. falciparum malaria; 2/22 (9.1% [0-21%]) were RIII resistant. Doxycycline cured 11/17 (64.7% [42.0-87.4%]) patients, and chloroquine 4/20 (20% [2.5-37.5%]). Against P. vivax, chloroquine/doxycycline cured (S) 12/17 (70.6% [48.9-92.2%]) patients, doxycycline 4/12 (33.3% [6.6-59.9%]), and chloroquine 5/17 (29.4% [7.7-51.1%]). Chloroquine/doxycycline was effective against P. falciparum but only modestly effective against P. vivax. These findings support the use of chloroquine/doxycycline as an inexpensive alternative to mefloquine for treating chloroquine-resistant P. falciparum but not chloroquine-resistant P. vivax in this setting.     

Correlation of in vivo-resistance to chloroquine and allelic polymorphisms in Plasmodium falciparum isolates from Uganda

The efficacy of chloroquine in the treatment of uncomplicated falciparum malaria in Africa is heavily compromised by high levels of drug resistance. The occurrence of active site mutations in the Plasmodium falciparum multi drug resistance-gene 1 (pfmdr1) has been associated with development of resistance to chloroquine. This study investigates the occurrence of several mutations at codons 86, 1042 and 1246 of the pfmdr1-gene in infected blood samples taken from Ugandan children before treatment with chloroquine and their relationship to clinical and parasitological resistance. Even though a clear association of CQR to one certain pfmdr1 single point mutation could not be substantiated, the frequency of resistance was consistently higher for samples revealing any of the mutations than among wild type samples, and 90% of the clinically resistant samples did present a mutation. Thus detection of these allelic pfmdr1 polymorphisms is not a decisive factor for prediction of clinical chloroquine resistance, but an interplay of the different mutations with unknown cofactors is to be assumed and the possible role of other genetic alterations remains to be investigated.     

Plasmodium falciparum in Kenya: high prevalence of drug-resistance-associated polymorphisms in hospital admissions with severe malaria in an epidemic area.

During an epidemic of Plasmodium falciparum malaria in Chogoria, Kenya, P. falciparum DNA was collected from 24 cases of severe malaria admitted to hospital for parenteral quinine treatment. These patients had all failed first- (chloroquine) and second-line (sulfadoxine-pyrimethamine or amodiaquine) drug treatments. Twenty-two (92%) of the 24 patients sampled carried parasites with the (Asn)86(Tyr) point mutation in the pfmdr1 gene (chromosome 5), 20 (83%) had an (Asp)1246(Tyr) mutation and 18 (82%) had both of these mutations. These alleles are both reported to be associated with chloroquine-resistance. Polymorphisms in the cg2 gene (chromosome 7) are also associated with chloroquine resistance, and 18 (75%) of the 24 parasite samples each had the cg2 and pfmdr1 polymorphisms. These 18 samples also had the mutations associated with resistance to pyrimethamine and sulfadoxine: (Asn)51(Ile), (Cys)59(Arg) and (Ser)108(Asn) of gene dhfr (chromosome 4) and (Ala)437(Gly) and (Lys)540(Glu) of dhps (chromosome 8), respectively. Genotyping of the parasites from all 24 patients revealed extensive diversity in the sequences for the merozoite surface antigens (MSA-1 and MSA-2) and the glutamate-rich protein (GLURP) and indicated that each sample contained more than one parasite clone. Although samples from non-admitted malaria cases were not available, it appears that drug resistance may have played an important role in the development of severe malaria in this epidemic.     

Genetics of drug-resistant Plasmodium falciparum malaria in the Venezuelan state of Bolivar

The state of Bolivar in Venezuela experiences episodic outbreaks of multidrug-resistant Plasmodium falciparum malaria. We obtained P. falciparum-infected blood samples in Bolivar in 1998-2000, and performed molecular assays for mutations conferring resistance to the antifolate combination of sulfadoxine-pyrimethamine (SP) and to chloroquine. All infections carried the dihydrofolate reductase (dhfr) S108A and N51I mutations, and 45% of the infections had the dhfr C50R mutation, which has been implicated in mid-level resistance to SP. Two dihydropteroate synthase (dhps) mutations also involved in SP resistance, A581G and K540E, were detected in 90% and 67% of the samples, respectively. The dhfr 1164L mutation, which confers high-level resistance, was not identified. The P. falciparum chloroquine resistance transporter (pfcrt) K76T mutation, which is critical for chloroquine resistance, was found in 167 of 168 infections. Six dhfr/dhps allelotypes and four pfcrt-resistant alleles were observed. Their interrelationships suggest a semi-clonal propagation of P. falciparum malaria in Bolivar, and an invasion of multi-resistant pathogens from Brazil. Despite national restrictions on the use of SP and chloroquine, genotypic resistance to these therapies remains widespread in Bolivar.     

Strong association, but incomplete correlation, between chloroquine resistance and allelic variation in the pfmdr-1 gene of Plasmodium falciparum isolates from India

The increasing prevalence of chloroquine-resistant Plasmodium falciparum has complicated the control of falciparum malaria. It has been suggested that point mutations at nucleotide positions 754, 1049, 3598, 3622 and 4234 in the parasite's pfmdr-1 gene are associated with such resistance, although this is a matter of controversy. Eighteen chloroquine-sensitive and 22 resistant isolates of P. falciparum from India were investigated, to examine the role of the pfmdr-1 gene in the resistance, and to determine whether any of the point mutations could be used as a marker for the rapid identification of the chloroquine-resistant strains. As this investigation failed to reveal an explicit association between allelic variation in the pfmdr-1 gene and chloroquine resistance, the use of point mutations to identify the resistant strains does not appear feasible.