BackgroundMalaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes.MethodsEight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru.FindingsEight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes.ConclusionsThis is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries.

Background: Several strategies are currently deployed in many countries in the tropics to strengthen malaria control toward malaria elimination. To measure the impact of any intervention, there is a need to detect malaria properly. Mostly, decisions still rely on microscopy diagnosis. But sensitive diagnosis tools enabling to deal with a large number of samples are needed. The molecular detection approach offers a much higher sensitivity, and the flexibility to be automated and upgraded.

Background: The combination of artesunate and mefloquine was introduced as the national firstline treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy.

BackgroundMalaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia.MethodsA new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy.Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections.ResultsThe sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%).ConclusionsThe rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.

SummaryArtesunate and mefloquine combination treatment has been used since 2000 in Cambodia as the firstline drug for the treatment of uncomplicated falciparum malaria. In order to assess its efficacy and safety, the national malaria control programme conducted 14 therapeutic efficacy studies with the drug combination between 2001 and 2004 at nine sites. In 2001 and 2002, co-blister packs of artesunate and mefloquine were used, whereas in 2003 and 2004, drugs were given individually from a bulk pack at a total dose of 12 mg/kg of artesunate and 25 mg/kg of mefloquine over 3 days. A total of 1025 patients were enrolled over the 4 years and 977 were follow-up during the period of 28 days. The PCR-corrected cure rates ranged from 85.7% to 100% with an overall cure rate of 95.8% (920/960). The studies in 2002 showed also that co-blister packs used on the basis of age and not on the basis of weight could lead to underdosed regimens but without any detectable effect on the treatment outcome. The follow-up period was extended from 28 to 42 days in three sites in 2004. A total of 219 among 255 were follow-up until day 42. The cure rate decreased but not significantly from 90.1% (73/81) with 28 days follow-up to 79.3% (46/58) with 42 days follow-up in Pailin, whereas the cure rate remained at 100% in the two other sites. Side effects were common, especially dizziness, but were mild and transient and patients recovered without any medical intervention.