The potential use of methotrexate in the treatment of falciparum malaria: in vitro assays against sensitive and multidrug-resistant falciparum strains.

The anti-malarial activity of the anti-cancer drug methotrexate against chloroquine-sensitive T9-96 and the multidrug-resistant K1 strains of Plasmodium falciparum was assessed in vitro. Mean IC50 values of 0.32 +/- 0.05 nM and 48.02 +/- 4.40 nM were obtained for T9-96 and K1, respectively, indicating methotrexate's high potency against both sensitive and resistant P. falciparum strains in vitro. Our results suggest that methotrexate is potentially effective against falciparum malaria in short-term, low-dose regimens, minimizing the risk of toxicity. This, along with the practical advantages of methotrexate, warrants the clinical investigation of methotrexate in human cases of falciparum malaria.

[Presence of the double pfmdr1 mutation 86Tyr and 1246 Tyr in clones of a chloroquine-resistant west African isolate of Plasmodium falciparum]. / Detecção da mutação dupla 86TYR e 1246TYR no gene pfmdr1 em clones de uma amostra de Plasmodium falciparum da África Ocidental, resistente à cloroquina.

Isolates of Plasmodium falciparum from three areas of West Africa were recovered from cryopreservation and their chloroquine-sensitivity were determined in vitro. Of the 90 samples studied, 60 were from Guinea-Bissau (30Resistant/30Sensitive), 15 were from S. Tomé and Príncipe (11Resistant/4Sensitive) and 15 were from Angola (11Resistant/4Sensitive). All the isolates were sensitive to mefloquine. Using the polymerase chain reaction/restriction fragment length polymorphism technique (PCR/RFLP) it was possible to detect two mutations in the pfmdr1 gene, often associated with chloroquine-resistance. 66% of the samples from Guiné-Bissau showed a correlation with chloroquine-resistance while 73% of the samples from São Tomé and Angola altogether had the 86Tyr mutation. The present study on West African isolates and clones showed, for the first time, the presence of a double point mutation in the pfmdr1 gene one being found, up to now, only in South America isolates of Plasmodium falciparum.

In vitro activity of nitazoxanide and related compounds against isolates of Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis.

The activities of the N-(nitrothiazolyl) salicylamide nitazoxanide and its metabolite tizoxanide were compared with metronidazole in vitro in microplates against six axenic isolates of Giardia intestinalis. Tizoxanide was eight times more active than metronidazole against metronidazole-susceptible isolates and twice as active against a resistant isolate. In 10 axenic isolates of Entamoeba histolytica, while tizoxanide was almost twice as active as metronidazole against more susceptible isolates, it was more than twice as active against less susceptible isolates. Fourteen metronidazole-susceptible isolates of Trichomonas vaginalis were 1.5 times more susceptible to tizoxanide, which was nearly five times as active against resistant isolates. Two highly metronidazole-resistant isolates retained complete susceptibility to tizoxanide, and one moderately resistant isolate showed reduced susceptibility. In all three organisms, nitazoxanide results paralleled those of tizoxanide. Analogues lacking the reducible nitro-group had similar low activities against susceptible G. intestinalis, E. histolytica and T. vaginalis, indicating that nitro-reduction and free radical production was a probable mode of action. Nitazoxanide and its metabolite tizoxanide are more active in vitro than metronidazole against G. intestinalis, E. histolytica and T. vaginalis. Although, like metronidazole, they depend on the presence of a nitro-group for activity, they retain some activity against metronidazole-resistant strains, particularly of T. vaginalis. The results suggest that resistance mechanisms for metronidazole can be bypassed by nitazoxanide and tizoxanide.

Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria.

In view of the recent discovery (Molecular Cell 6, 861-871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology, 119, 343-348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine.

Can pretreatment screening for dhps and dhfr point mutations in Plasmodium falciparum infections be used to predict sulfadoxine-pyrimethamine treatment failure?

This study examines the relationship between malaria treatment failure after sulfadoxine-pyrimethamine (S-P) chemotherapy and presence of mutations in the Plasmodium falciparum dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes (associated with resistance in vitro to S and P) before treatment. In Kenya, 38 malaria patients in a holoendemic area, and 21 in an epidemic area, participated in the trial in 1997-98. In the 2 areas, drug failure occurred in 76% and 75% of cases where any mutation in dhfr was seen (positive predictive values 76% and 75%: P = 0.003 and 0.008) and an identical association was seen with dhfr Asn-108. In the holoendemic area all occurrences of > or = 2 mutations in dhfr predicted drug failure. Only 3 instances were seen in the epidemic focus, but treatment failed in all. Only in the epidemic focus, 7 (88%) of 8 occurrences of > or = 1 mutations in dhps, and all occurrences of the Gly-437 allele of dhps, predicted failure. Association between mutations in dhps and mutations in dhfr was noted in the combined sites, irrespective of outcome. Although this makes the relationship of combined dhfr and dhps mutations to failure more difficult to interpret, it nevertheless supports S-P selection acting on both genes. In the holoendemic site, treatment success increased with age. In this location, acquired immunity may mask the impact of mutations in dhps, since sulfadoxine is a less effective treatment than pyrimethamine.

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.

Co-trimoxazole compared with sulfadoxine-pyrimethamine in the treatment of uncomplicated malaria in Kenyan children.

Sulfadoxine-pyrimethamine (SP) and co-trimoxazole were both effective in reducing fever, clearing parasitaemia and improving anaemia in children aged < 5 years with uncomplicated malaria in 2 Kenyan endemic sites, Oyugis in the west and Tiwi on the coast. We compared the efficacy of these 2 regimens (in May-July 1998) by evaluating clinical and parasitological responses over 14 days. The combined incidence of parasitological failure for the combined sites for co-trimoxazole was 14/123 (11%) and for SP 23/145 (16%) (RR 0.72, 95% confidence interval [CI] 0.31-1.46, P = 0.289). The 14-day clinical failure rate for the combined sites for co-trimoxazole was 4/123 (3.3%), and for SP 8/145 (5.5%), (RR 1.69, 95% CI 0.91-3.15, P = 0.129). The results indicate that the risk of treatment failure for the 2 regimens was similar. The antimalarial use of co-trimoxazole in uncomplicated malaria needs further investigation, since the 10-12-h elimination half-life of both components should reduce selective pressure for resistance. In addition, use of a 2-day high-dose course, tested previously, requires further study to demonstrate its efficacy.

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.

Sequence diversity of serine repeat antigen gene exon II of Plasmodium falciparum in worldwide collected wild isolates.

Field isolates of Plasmodium falciparum collected from endemic areas of Southeast Asia, Solomon Islands, tropical African countries and Brazil were analyzed for the genetic diversity of the exon II of serine repeat antigen gene (SERA) by sequencing of genomic DNA. Of sixty-nine isolates, as compared to the reported FCR3, K1 and Honduras-1 types of exon II sequences, 5, 9 and 20 new allelic forms were found in 23 isolates of the FCR3 type, 36 of the K1 type and 10 of the Honduras-1 type. A group of novel non-synonymous substitutions, 4 new insertions and 3 new deletions of octamer units were found in the octamer repeat region (OR) of the exon II, and most of them clustered within a 40-residues domain. An octamer "SNPVSSEP" revealed in the OR was confirmed as a new repeat unit. Based on the sequences of the serine repeat region (SR) of the exon II, the allelic forms of the Honduras-1 type were conjectured to be the recombinant forms between the K1 type and FCR3 type. The allelic forms of K1 type with less or more repeat serine residues in the serine stretch of the SR than the reported 21 serine residues had most of the variations in the OR. Moreover, a biased geographical distribution of allelic forms was observed. Isolates from African and Southeast Asian countries accounted for most of the new allelic forms (29/33). All of the three types were detected in Southeast Asia but none of the FCR3 type in Africa. One of two groups of FCR3 new allelic forms was found solely in Brazil while another was mainly in Solomon Islands.

Association of cg2 and pfmdr1 genotype with chloroquine resistance in field samples of Plasmodium falciparum from Nigeria.

This study examines polymorphisms in 2 genes (pfmdr1 and cg2), which have been associated with resistance to chloroquine in Plasmodium falciparum, to determine their value as predictors of resistance status. Among field samples from children in Zaria, northern Nigeria, the Tyr-86 polymorphism in pfmdr1 and Ala-281 and the Dd2 kappa repeat of cg2, were significantly associated. In 8 samples classified resistant by the micro-in vitro test, or, where this failed, by in vivo trial, 7 showed the cg2 Dd2 type kappa repeat, and 6 of these had both the Ala-281 allele and the pfmdr1 Tyr-86 allele. In 26 chloroquine-sensitive samples, none had this combination of 3 polymorphisms (P = 0.00002). This indicates 75% sensitivity and 100% specificity in detection of resistance and shows a positive predictive value for resistant infections of 100%. The negative predictive value, because of sensitivity less than 100%, would depend on the prevalence of resistance. Where prevalence of resistance is approx. 21% as in Zaria, the negative predictive value would be 94%, while in Gabon, with a prevalence of ca 73% it would be 60%. The use of (cg2: Ala-281, Dd2 kappa. pfmdr1: Tyr-86) genotype detection as a predictive epidemiological tool to examine the distribution of chloroquine-resistance in parts of Africa is therefore possible. The sensitivity of detection of resistant strains still requires improvement.

Allele-specific, nested, one tube PCR: application to Pfmdr1 polymorphisms in Plasmodium falciparum.

An allele-specific, one tube PCR for the sensitive and reliable detection of point mutations in Plasmodium falciparum DNA is described. Design of specific internal primers and optimization of the PCR is simple, and the procedure is robust and sensitive. Single nucleotide polymorphisms at codons 184, 1034, 1042 and 1246 of the P, falciparum multidrug resistance gene Pfmdr1, were examined in 6 laboratory isolates, to validate the technique.

Plasmodium falciparum: in vitro chloroquine susceptibility and allele-specific PCR detection of Pfmdr1 Asn86Tyr polymorphism in Lambarene, Gabon.

Plasmodium falciparum resistance to chloroquine has been described in many parts of the world particularly in Africa where malaria is endemic. High levels of chloroquine resistance in our study area, Lambarene-Gabon, has led to the use of an alternative regimen for treatment and prevention of P. falciparum infection. In this study, we examined the in vitro chloroquine sensitivity of 15 isolates from this area and assessed the prevalence of a putative chloroquine resistance associated Pfmdr1 polymorphism (Asn86Tyr) using a novel allele-specific polymerase chain reaction (PCR). Only 4 of the isolates examined were chloroquine sensitive. The allele-specific PCR shows that all 15 isolates carried the variant (86Tyr) codon. Eleven of these were resistant to chloroquine suggesting a 73% agreement between chloroquine resistance phenotype and the point mutation. This molecular marker was examined in a further 73 Gabonese isolates, where 58 (79.5%) showed 86Tyr and 15 (20.5%) showed 86Asn. In all, 4 (4.5%) of the 88 isolates assessed carry both mutant and wild-type codons, suggesting mixed parasite populations. The incomplete agreement found between chloroquine resistance phenotype and Pfmdr1 (86Tyr) polymorphism would support the view that other genetic factors as well as Pfmdr1 may be involved in chloroquine resistance. While our results suggest a high prevalence of 86Tyr polymorphism in Lambarene, the Asp1246Tyr polymorphism (a point mutation which to date has only been associated with South American P. falciparum) seems to be absent in our study area.

Pfmdr1 Asn1042Asp and Asp1246Tyr polymorphisms, thought to be associated with chloroquine resistance, are present in chloroquine-resistant and -sensitive Brazilian field isolates of Plasmodium falciparum.

Parasite resistance to antimalarial drugs, particularly chloroquine, is the most disturbing problem of malaria chemotherapy. There is evidence that the codon 86Tyr polymorphism of the Pfmdr1 gene is associated with chloroquine resistance in West African Plasmodium falciparum. The association of this and four other coding alterations of the Pfmdr1 gene with chloroquine resistance has not been extensively investigated in South American isolates. In this study, we examined 51 Brazilian P. falciparum isolates for the presence or absence of Asn86Tyr, Asn1042Asp, and Asp1246Tyr polymorphisms. While these isolates were all sensitive in vitro to mefloquine, amodiaquine, and quinine, only 2 (4%) were chloroquine-sensitive. The findings reported here provide the first observations of this kind on a large number of field parasite samples from South America. We show that in vitro chloroquine-resistant and -sensitive strains carry the Asn1042Asp and Asp1246Tyr polymorphisms and provide support for earlier suggestions that Asn86Tyr may be rare or absent in South American P. falciparum.

Simplified diagnosis of malaria infection: GFM/PCR/ELISA a simplified nucleic acid amplification technique by PCR/ELISA.

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

Antimalarial drug response of Plasmodium falciparum from Zaria, Nigeria.

The sensitivity of Zaria strains of Plasmodium falciparum to chloroquine, mefloquine, quinine and sulphadoxine/pyrimethamine was investigated 5 years after the appearance of in vivo/in vitro chloroquine resistance in urban Zaria. Infections in 36/43 children (83.7%) treated with chloroquine were sensitive while those in 7 (16.3%) were resistant. 8/13 isolates cultured (61.5%) were sensitive in vitro to chloroquine and 5 (38.5%) were resistant. Of the cultured isolates, 13/13 (100%), 12/13 (92.3%) and 5/7 (71.4%) showed mefloquine, quinine and sulphadoxine/pyrimethamine sensitivity, respectively. The results confirmed chloroquine and sulphadoxine/pyrimethamine resistance in urban Zaria and revealed emerging quinine resistance. Resistance to chloroquine and sulphadoxine/pyrimethamine is at RI level and chloroquine should continue to be the first-line drug for the treatment and prevention of P. falciparum infection in the Zaria area of northern Nigeria. We suggest that, while quinine serves as second-line drug, mefloquine should be reserved for infections resistant to chloroquine, quinine and sulphadoxine/pyrimethamine.