I.S. Adagu

Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria

We report the first in vitro and genetic confirmation of Malarone® (GlaxoSmithKline; atovaquone and proguanil hydrochloride) resistance in Plasmodium falciparum acquired in Africa. On presenting with malaria two weeks after returning from a 4-week visit to Lagos, Nigeria without prophylaxis, a male patient was given a standard 3-day treatment course of Malarone®. Twenty-eight days later the parasitaemia recrudesced. Parasites were cultured from the blood and the isolate (NGATV01) was shown to be resistant to atovaquone and the antifolate pyrimethamine. The cytochrome b gene of isolate NGATV01 showed a single mutation, Tyr268Asn which has not been seen previously.

Modified Fixed-Ratio Isobologram Method for Studying In Vitro Interactions between Atovaquone and Proguanil or Dihydroartemisinin against Drug-Resistant Strains of Plasmodium falciparum

ABSTRACT A modified fixed-ratio isobologram method for studying the in vitro interactions between antiplasmodial drugs is described. This method was used to examine the interactions between atovaquone, proguanil, and dihydroartemisinin. The interaction between atovaquone and proguanil was synergistic against atovaquone-sensitive strains K1 and T996; however, there was a loss of synergy against atovaquone-resistant strain NGATV01 isolated after Malarone (the combination of atovaquone and proguanil) treatment failure. While the interaction between atovaquone and dihydroartemisinin was indifferent against isolate NGATV01, the interaction displayed indifference tending toward antagonism against the atovaquone-sensitive strains tested. The relevance of in vitro interactions to in vivo treatment is discussed.

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.

The relationship of physico-chemical properties and structure to the differential antiplasmodial activity of the cinchona alkaloids

BackgroundThe 8-amino and 9-hydroxy substituents of antimalarial cinchona alkaloids have the erythro orientation while their inactive 9-epimers are threo. From the X-ray structures a 90° difference in torsion angle between the N1-H1 and C9-O12 bonds in the two series is believed to be important. In order to kill the malaria parasite, alkaloids must cross the erythrocyte and parasite membranes to accumulate in the acid digestive vacuole where they prevent detoxication of haematin produced during haemoglobin breakdown.MethodsIonization constants, octanol/water distribution and haematin interaction are examined for eight alkaloids to explain the influence of small structural differences on activity.ResultsErythro isomers have a high distribution ratio of 55:1 from plasma to the erythrocyte membrane, while for the more basic threo epimers this is only 4.5:1. This gives an increased transfer rate of the erythro drugs into the erythrocyte and thence into the parasite vacuole where their favourable conformation allows interaction with haematin, inhibiting its dimerization strongly (90 ± 7%) and thereby killing the parasite. The threo compounds not only enter more slowly but are then severely restricted from binding to haematin by the gauche alignment of their N1-H1 and C9-O12 bonds. Confirmatory molecular models allowed measurement of angles and bond lengths and computation of the electronic spectrum of a quinine-haematin complex.ConclusionDifferences in the antiplasmodial activity of the erythro and threo cinchona alkaloids may therefore be attributed to the cumulative effects of lipid/aqueous distribution ratio and drug-haematin interaction. Possible insights into the mechanism of chloroquine-resistance are discussed.

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.

Lysosomes and drug resistance in malaria

It is now clear that drugs must be designed that will simultaneously block the integral lysosomal membrane protein (PfCRT)-resistance mechanism and attack the haematin target. That these are not contradictory aims is shown by the relative success of amodiaquine mefloquine and the artemisinins. X-ray crystallography of drug-haematin interaction has not yet been reported and together with more functional studies and structural analysis of PfCRT should be urgently pursued to give a template for much needed new drugs. Haematin remains a key target for antimalarial drug design. Meanwhile in developing countries attempts must continue with the help of international agencies to solve the problems related to cash and infrastructure of access to treatment. (excerpt)

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.

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.

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.

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.