Post-treatment haemolysis in African children with hyperparasitaemic falciparum malaria; a randomized comparison of artesunate and quinine.

BACKGROUND: Parenteral artesunate is the treatment of choice for severe malaria. Recently, haemolytic anaemia occurring 1 to 3 weeks after artesunate treatment of falciparum malaria has been reported in returning travellers in temperate countries. METHODS: To assess these potential safety concerns in African children, in whom most deaths from malaria occur, an open-labelled, randomized controlled trial was conducted in Kinshasa, Democratic Republic of Congo. 217 children aged between 6 months and 14 years with acute uncomplicated falciparum malaria and parasite densities over 100,000/µL were randomly allocated to intravenous artesunate or quinine, hospitalized for 3 days and then followed for 42 days. RESULTS: The immediate reduction in haemoglobin was less with artesunate than with quinine: median (IQR) fall at 72 h 1.4 g/dL (0.90-1.95) vs. 1.7 g/dL (1.10-2.40) (p = 0.009). This was explained by greater pitting then recirculation of once infected erythrocytes. Only 5% of patients (in both groups) had a ≥ 10% reduction in haemoglobin after day 7 (p = 0.1). One artesunate treated patient with suspected concomitant sepsis had a protracted clinical course and required a blood transfusion on day 14. CONCLUSIONS: Clinically significant delayed haemolysis following parenteral artesunate is uncommon in African children hospitalised with acute falciparum malaria and high parasitaemias. TRIAL REGISTRATION: ClinicalTrials.gov ; Identifier: NCT02092766 (18/03/2014).

Randomized comparison of the efficacies and tolerabilities of three artemisinin-based combination treatments for children with acute Plasmodium falciparum malaria in the Democratic Republic of the Congo.

An open-label, randomized controlled trial was carried out in 2011-2012 in the Democratic Republic of the Congo to test the efficacy, safety, and tolerability of the artemisinin-based combination treatments dihydroartemisinin-piperaquine, amodiaquine-artesunate, and artemether-lumefantrine. Six hundred eighty-four children aged 3 to 59 months with uncomplicated Plasmodium falciparum malaria were randomly allocated to each study arm. Children were hospitalized for 3 days, given supervised treatment, and followed up weekly for 42 days. All regimens were well tolerated and rapidly effective. The median parasitemia clearance half-life was 2.2 h, and half-lives were similar between arms (P=0.19). The PCR-uncorrected cure rates by day 42 were 73.0% for amodiaquine-artesunate, 70.2% for artemether-lumefantrine, and 86.3% for dihydroartemisinin-piperaquine (P=0.001). Early treatment failure occurred in three patients (0.5%), one in each arm. The PCR-corrected cure rates were 93.4% for amodiaquine-artesunate, 92.7% for artemether-lumefantrine, and 94.3% for dihydroartemisinin-piperaquine (P=0.78). The last provided a longer posttreatment prophylactic effect than did the other two treatments. The day 7 plasma concentration of piperaquine was below 30 ng/ml in 47% of the children treated with dihydroartemisinin-piperaquine, and the day 7 lumefantrine concentration was below 280 ng/ml in 37.0% of children who received artemether-lumefantrine. Thus, although cure rates were all satisfactory, they could be improved by increasing the dose. (This study has been registered with the International Standard Randomized Controlled Trial Number Register [www.isrctn.org] under registration no. ISRCTN20984426.).

Malaria diagnosis under field conditions in the Venezuelan Amazon.

To improve practical, accurate diagnosis of malaria in the Amazon rainforest of Venezuela, two rapid diagnostic tests (RDT) (OptiMAL-IT) and FalciVax) and a laboratory light microscope, used in the field with a battery-operated head lamp as an external light source, were evaluated against the standard laboratory microscope procedure for malaria detection. One hundred and thirty-six Yanomami patients were studied for the presence of malaria parasites. Thirty-three patients (24%) were positive for malaria (Plasmodium falciparum, P. vivax, P. malariae). Twenty-one (64%) of the positive patients had <100 parasites/microl. Both RDTs showed poor sensitivity (24.2% for OptiMAL-IT) and 36.4% for FalciVax) but good specificity (99% both for OptiMAL-IT) and FalciVax). Field and laboratory microscopy showed sensitivities of 94% and 91%, respectively. The kappa coefficient was 0.90, indicating a high agreement between field and laboratory microscopy. We conclude that (i) adequate slide reading cannot be substituted by either of the two RDTs in the Venezuelan Amazon and (ii) the use of a light source such as that described above makes slide reading more feasible than hitherto in remote areas without electricity.

Anaemia and malaria in Yanomami communities with differing access to healthcare.

Inequitable access to healthcare has a profound impact on the health of marginalised groups that typically suffer an excess burden of infectious disease morbidity and mortality. The Yanomami are traditionally semi-nomadic people living in widely dispersed communities in Amazonian Venezuela and Brazil. Only communities living in the vicinity of a health post have relatively constant access to healthcare. To monitor the improvement in the development of Yanomami healthcare a cross-sectional survey of 183 individuals was conducted to investigate malaria and anaemia prevalence in communities with constant and intermittent access to healthcare. Demographic and clinical data were collected. Malaria was diagnosed by microscopy and haemoglobin concentration by HemoCue. Prevalence of malaria, anaemia, splenomegaly, fever and diarrhoea were all significantly higher in communities with intermittent access to healthcare (anaemia 80.8% vs. 53.6%, P<0.001; malaria 18.2% vs. 6.0%, P=0.013; splenomegaly 85.4% vs.12.5%, P<0.001; fever 50.5% vs. 28.6%, P=0.003; diarrhoea 30.3% vs.10.7% P=0.001). Haemoglobin level (10.0 g/dl vs. 11.5 g/dl) was significantly associated with access to healthcare when controlling for age, sex, malaria and splenomegaly (P=0.01). These findings indicate a heavy burden of anaemia in both areas and the need for interventions against anaemia and malaria, along with more frequent medical visits to remote areas.

A randomised trial to assess the efficacy and safety of chlorproguanil/dapsone + artesunate for the treatment of uncomplicated Plasmodium falciparum malaria.

We tested the efficacy and safety of chlorproguanil/dapsone co-administered with artesunate (CD+A) for the treatment of uncomplicated Plasmodium falciparum malaria in children compared with amodiaquine+sulfadoxine/pyrimethamine (AQ+SP) at two different sites in Rwanda. The trial was open label and 800 patients were randomly assigned to AQ+SP (n=400) or CD+A (n=400). Patients were hospitalised for 3 days and then followed-up weekly until Day 28 after treatment. Clinical and parasitological outcomes were recorded. Results showed that neither treatment was adequately efficacious. At one site, the adequate clinical and parasitological response (ACPR), PCR-adjusted, was 73.3% in the CD+A arm and 87.8% in the AQ+SP arm (P<0.001), and at the second site the ACPR, PCR-adjusted, was 70.5% in the CD+A arm and 38.1% in the AQ+SP arm (P<0.001). The combination CD+A is considered an alternative to, or replacement for, SP in Africa because CD has been shown to be effective in patients for whom SP treatment has failed and, with its short half-life, it is expected to exert less selection pressure for resistant parasites than SP. However, the results of this trial indicate that in an area of high SP resistance, CD+A may not be the best choice.

A randomised trial to assess the safety and efficacy of artemether-lumefantrine (Coartem) for the treatment of uncomplicated Plasmodium falciparum malaria in Rwanda.

Coartem is a fixed-dose combination of artemether-lumefantrine that, given in six doses, provides effective treatment for children with uncomplicated Plasmodium falciparum infection in areas with highly endemic and multidrug-resistant malaria. In Rwanda since 2001, amodiaquine+sulfadoxine-pyrimethamine (AQ+SP) has been the first-line treatment, but resistance to this combination has rapidly emerged and spread. Coartem was considered as a possible alternative, and a randomised, open-label, clinical trial to test its safety, tolerability and efficacy was carried out in 2004-2005. Five hundred children aged 12-59 months with uncomplicated P. falciparum malaria were randomly allocated to AQ+SP or Coartem. Patients were followed up until day 28 after treatment. Adverse events and clinical and parasitological outcomes were recorded. Adequate clinical and parasitological response (ACPR) was significantly higher in children treated with Coartem than in those treated with AQ+SP: the PCR-adjusted 28-day ACPR was 96.68% for Coartem and 79.35% for AQ+SP. Both treatments rapidly cleared parasitaemia and fever, although parasite clearance was significantly faster in children treated with Coartem. Mean packed cell volume increased in all patients, with no significant differences between treatments. Coartem proved to be more efficacious than AQ+SP, with a good safety and tolerability profile.

Origin of Plasmodium falciparum malaria is traced by mitochondrial DNA.

The origin and geographical spread of Plasmodium falciparum is here determined by analysis of mitochondrial DNA sequence polymorphism and divergence from its most closely related species P. reichenowi (a rare parasite of chimpanzees). The complete 6 kb mitochondrial genome was sequenced from the single known isolate of P. reichenowi and from four different cultured isolates of P. falciparum, and aligned with the two previously derived P. falciparum sequences. The extremely low synonymous nucleotide polymorphism in P. falciparum (pi=0.0004) contrasts with the divergence at such sites between the two species (kappa=0.1201), and supports a hypothesis that P. falciparum has recently emerged from a single ancestral population. To survey the geographical distribution of mitochondrial haplotypes in P. falciparum, 104 isolates from several endemic areas were typed for each of the identified single nucleotide polymorphisms. The haplotypes show a radiation out of Africa, with unique types in Southeast Asia and South America being related to African types by single nucleotide changes. This indicates that P. falciparum originated in Africa and colonised Southeast Asia and South America separately.

The kdr pyrethroid resistance gene in Anopheles gambiae: tests of non-pyrethroid insecticides and a new detection method for the gene.

The organophosphate pirimiphos-methyl and the carbamate carbosulfan were evaluated in comparison to the pyrethroid alphacypermethrin and the 'near-pyrethroid' etofenprox against pyrethroid resistant Anopheles gambiae and Culex spp. in an experimental hut station located in central Côte d'Ivoire. Bednets were impregnated with the above mentioned compounds and randomly allocated to the huts. On 40 consecutive mornings, after sleepers had occupied the huts overnight, mosquitoes were collected from the huts, identified and scored as live or dead (including delayed mortality). An. gambiae s.l. that had been collected were tested for the presence of the kdr allele in heterozygous or homozygous form. Both non-pyrethroid treatments caused very high mortality, whereas mortality with alpha-cypermethrin and etofenprox generally did not differ from the levels observed with untreated control nets in this experiment. The nets had holes cut in them and there was considerable bloodfeeding on the sleepers, which was only significantly reduced for An. gambiae by carbosulfan and alpha-cypermethrin. PCR genotyping suggested that there was selection for the kdr resistance allele by the pyrethroid treated nets. Organophosphates and carbamates may therefore present an alternative to be used on bednets especially in areas of pyrethroid resistance, but the safety of these insecticides will have to be carefully considered.

Comparison of the Paracheck-Pf test with microscopy, for the confirmation of Plasmodium falciparum malaria in Tanzania.

Paracheck-Pf is a rapid, qualitative immuno-assay for the detection of Plasmodium falciparum-specific histidine-rich protein-2 in samples of human blood. The assay has now been evaluated, against the usual 'gold standard', microscopy, using blood samples from 1655 individuals in five districts of Tanzania, four of which experience frequent malaria outbreaks. The aim was to verify whether Paracheck-Pf could be a reliable tool for the confirmation of malaria outbreaks in such areas. The overall measurements of the assay's performance were good, with a sensitivity of 90.0%, a specificity of 96.6%, a positive predictive value of 88.9%, and a negative predictive value of 97.0% (with an estimated malaria prevalence of 23.3%). There was, however, marked variation between the study districts, the assay's performance being relatively poor where the test had been stored for 12 months at room temperature (23.5+/-3.5 degrees C). The assay was easy to perform in the field and could clearly be a valuable tool in remote areas and in emergency situations, such as the early detection of malaria outbreaks. The cost of the assay (U.S.$0.62/test at the time of the present study) is sufficiently low that its routine use in the confirmation of P. falciparum malaria might also be cost-effective, particularly in areas where there are no facilities for microscopy and/or where the first-line treatment of malaria is based on relatively expensive artemisinin-based combinations.

Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR-RFLP.

For differential identification of sibling species in the Anopheles gambiae Giles complex (Diptera: Culicidae), including simultaneous separation of M and S molecular forms within An. gambiae Giles sensu stricto, we describe a PCR-RFLP method. This procedure is more efficient, faster and cheaper than those used before, so is recommended for large-scale processing of field-collected larval and adult specimens to be identified in malaria vector studies.

Variation in an intron sequence of the voltage-gated sodium channel gene correlates with genetic differentiation between Anopheles gambiae s.s. molecular forms.

We present the results of a geographical survey of genetic variation in Anopheles gambiae M and S molecular forms from ten African countries at Intron I of the voltage-gated sodium channel gene. We found two major haplotypes separated by a single mutational step, which cosegregate almost completely with the rDNA sites that identify M and S, consistent with previous estimates of strong reductions of gene flow between the two forms. We also report ten additional haplotypes stemming from the two major haplotypes, mostly present in single localities. The low levels of genetic variation found in this intronic region are discussed in light of a possible selective sweep. These findings offer additional elements to the ongoing debate on the amount of genetic differentiation and isolation between the two molecular forms and on their taxonomic status.

Molecular evidence of incipient speciation within Anopheles gambiae s.s. in West Africa.

We karyotyped and identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis Anopheles gambiae s.s. samples collected in several African countries. The data show the existence of two non-panmictic molecular forms, named S and M, whose distribution extended from forest to savannahs. Mosquitoes of the S and M forms are homosequential standard for chromosome-2 inversions in forest areas. In dry savannahs, S is characterized mainly by inversion polymorphisms typical of Savanna and Bamako chromosomal forms, while M shows chromosome-2 arrangements typical of Mopti and/or Savanna and/or Bissau, depending on its geographical origin. Chromosome-2 inversions therefore seem to be involved in ecotypic adaptation rather than in mate-recognition systems. Strong support for the reproductive isolation of S and M in Ivory Coast comes from the observation that the kdr allele is found at high frequencies in S specimens and not at all in chromosomal identical M specimens. However, the kdr allele does not segregate with molecular forms in Benin.

Experimental and molecular genetic analysis of the impact of pyrethroid and non-pyrethroid insecticide impregnated bednets for mosquito control in an area of pyrethroid resistance.

Experimental huts in Côte d'Ivoire were used to evaluate the pyrethroid alpha-cypermethrin, the non-ester pyrethroid etofenprox, the organophosphate pirimiphos-methyl and the carbamate carbosulfan on bednets against pyrethroid-resistant Anopheles gambiae Giles. To test for selection for the resistance gene by the treated nets, A. gambiae collected live or dead from the huts were kept and analysed for the presence of the kdr gene using a new polymerase chain reaction followed by sequence-specific oligonucleotide probing (PCR-SSOP) for kdr-genotyping. Deliberately holed bednets freshly treated with pirimiphos-methyl or carbosulfan caused over 90% kill of A. gambiae s.s. and Culex spp. However, the mortality with alpha-cypermethrin or etofenprox treated nets was similar to that with untreated nets. Bloodfeeding of A. gambiae s.s. on the sleepers under the nets was only significantly reduced by alpha-cypermethrin and carbosulfan. Tests of the residual activity of the bednets after seven months showed that pirimiphos-methyl had lost its efficacy while carbosulfan still performed well. Once again the pyrethroid treated nets gave similar results to the untreated nets. Selection for the kdr-allele by alpha-cypermethrin and etofenprox, but not by carbosulfan, was indicated by PCR-SSOP genotyping of mosquitoes. Thus carbamates such as carbosulfan, or organophosphates of longer persistence than pirimiphos-methyl and of low mammalian toxicity, would seem to be a promising alternative to be used on bednets, particularly in areas of pyrethroid resistance.

Comparative evaluation of carbosulfan- and permethrin-impregnated curtains for preventing house-entry by the malaria vector Anopheles gambiae in Burkina Faso.

Pyrethroid-impregnated bednets and curtains are widely employed to reduce the risk of malaria transmission, but pyrethroid-resistance is becoming more prevalent among malaria vector Anopheles mosquitoes (Diptera: Culicidae). As an alternative treatment for curtains, we assessed carbosulfan (a carbamate insecticide) in comparison with permethrin as the standard pyrethroid, against endophilic female mosquitoes of the Anopheles gambiae Giles complex in a village near Ouagadougou, Burkina Faso. The main criterion evaluated was the impact of curtains (hung inside windows, eaves and doorways) on the number of An. gambiae s.l. females active indoors at night. Light-traps were operated overnight (21.00-06.00 hours beside occupied untreated bednets) to sample mosquitoes in houses fitted with net curtains treated with carbosulfan 0.2 g ai/m2 or permethrin 1 g ai/m2 or untreated, compared with houses without curtains. The treated and untreated curtains significantly reduced the numbers of mosquitoes collected indoors, compared with houses without curtains. Carbosulfan-treated curtains had a highly significantly greater effect than permethrin-treated or untreated curtains, the scale of the difference being estimated as three-fold. However, there was no significant difference between the impact of untreated and permethrin-treated curtains on densities of An. gambiae s.l. trapped indoors. Samples of the An. gambiae complex comprised An. arabiensis Patton and both the S- and M-forms of An. gambiae Giles s.s. Susceptibility tests revealed some resistance to DDT and low frequencies of permethrin-resistance, insufficient to explain the poor performance of permethrin on curtains. Among survivors from the diagnostic dosage of permethrin were some specimens of all three members of the An. gambiae complex, but the kdr resistance mechanism was detected only in the S-form of An. gambiae s.s. Questions arising for further investigation include clarification of resistance mechanisms in, and foraging behaviour of, each member of the An. gambiae complex in this situation and the need to decide whether carbosulfan-treated curtains are acceptably safe for use to reduce risks of malaria transmission.

The pyrethroid knock-down resistance gene in the Anopheles gambiae complex in Mali and further indication of incipient speciation within An. gambiae s.s.

In Mali the Anopheles gambiae complex consists of An. arabiensis and Mopti, Savanna and Bamako chromosomal forms of An. gambiae s.s. Previous chromosomal data suggests a complete reproductive isolation among these forms. Sequence analysis of rDNA regions led to the characterization of two molecular forms of An. gambiae, named M-form and S-form, which in Mali correspond to Mopti and to Savanna/Bamako, respectively, while it has failed so far to show any molecular difference between Savanna and Bamako. The population structure of An. gambiae s.l. was analysed in three villages in the Bamako and Sikasso areas of Mali and the frequency of pyrethroid resistance of the knock-down resistance (kdr) type was calculated. The results show that the kdr allele is associated only with the Savanna form populations and absent in sympatric and synchronous populations of Bamako, Mopti and An. arabiensis. This is the first molecular indication of barriers to gene flow between the Bamako and Savanna chromosomal forms. Moreover, analyses of specimens collected in the Bamako area in 1987 show that the kdr allele was already present in the Savanna population at that time, and that the frequency of this allele has gradually increased since then.