Evaluation of a real-time PCR assay for malaria diagnosis in patients from Vietnam and in returned travellers.

Real-time PCR diagnosis of malaria has advantages over traditional microscopic methods, especially when parasitaemia is low and when dealing with mixed infections. We have developed a new real-time PCR with specific genes in each Plasmodium species present only in one copy to identify the four pathogenic Plasmodium spp. for humans. The sensitivity was less than 25 parasites/microl. No cross-hybridisation was observed with human DNA or among the four Plasmodium spp. Using LightCycler PCR and conventional microscopy, we compared the diagnosis of malaria in patients from Vietnam and in returned European travellers with suspicion of malaria. In patients from Vietnam with suspicion of malaria, one mixed infection was observed by PCR only; the remaining data (54 of 55 patients) correlated with microscopy. In 79 patients without symptoms, low parasitaemia was detected in 7 samples by microscopy and in 16 samples by PCR. In returned travellers, PCR results were correlated with microscopy for all four species in 48 of 56 samples. The eight discrepant results were resolved in favour of real-time PCR diagnosis. This new real-time PCR is a rapid, accurate and efficient method for malaria diagnosis in returned travellers as well as for epidemiological studies or antimalarial efficiency trials in the field.

Apparent absence of atovaquone/proguanil resistance in 477 Plasmodium falciparum isolates from untreated French travellers.

OBJECTIVES: We examined the atovaquone in vitro susceptibility and the cytochrome b (cytb) gene polymorphism of African Plasmodium falciparum isolates during the first years of atovaquone/proguanil use. PATIENTS AND METHODS: Between 1999 and 2004, we collected blood samples from French P. falciparum-infected patients returning from African countries. Atovaquone susceptibility was determined using an in vitro isotopic test and cytb genotyping was performed by restriction fragment length polymorphism analysis and sequencing. These results were analysed according to the clinical response to atovaquone/proguanil treatment. RESULTS: No in vitro atovaquone resistance (IC50 > 1900 nM) and no cytb mutation leading to the Y268S substitution were detected among 477 unexposed African P. falciparum isolates. Eight cytb polymorphisms were found outside the ubiquinone reduction site by sequencing the entire gene of 270 isolates. One atovaquone/proguanil treatment failure was documented; the post-treatment isolate had an atovaquone susceptibility of 8230 nM and the Ser268 Cytb change; the pre-treatment isolate, obtained 4 weeks previously, was Cytb Tyr268 (wild-type). CONCLUSIONS: No atovaquone/proguanil resistance was detected by phenotyping or genotyping among 477 unexposed African P. falciparum isolates. Atovaquone/proguanil-resistant parasite was detectable only in the post-treatment isolate from a treatment failure.

Polymorphism in plasmodium falciparum drug transporter proteins and reversal of in vitro chloroquine resistance by a 9,10-dihydroethanoanthracene derivative.

BG958 reverses resistance in chloroquine-resistant isolates from different countries. Five mutations in the Plasmodium falciparum crt (pfcrt) gene resulting in the amino acid changes K76T, M74I, N75E, A220S, and R371I are systematically identified in resistance-reversed Asian, African, and Brazilian parasites which possess the pfcrt (CIET) haplotype. In combination with BG958, the activity of chloroquine is increased in parasites with the N86Y mutation in pfmdr1.