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High mutation rate in mammalian mitochondrial DNA generates a highly divergent pool of alleles even within species that have dispersed and expanded in size recently. Phylogenetic analysis of 277 human mitochondrial genomes revealed a significant (P < 0.01) excess of rRNA and nonsynonymous base substitutions among hotspots of recurrent mutation. Most hotspots involved transitions from guanine to adenine that, with thymine-to-cytosine transitions, illustrate the asymmetric bias in codon usage at synonymous sites on the heavy-strand DNA. The mitochondrion-encoded tRNAThr varied significantly more than any other tRNA gene. Threonine and valine codons were involved in 259 of the 414 amino acid replacements observed. The ratio of nonsynonymous changes from and to threonine and valine differed significantly (P = 0.003) between populations with neutral (22/58) and populations with significantly negative Tajima's D values (70/76), independent of their geographic location. In contrast to a recent suggestion that the excess of nonsilent mutations is characteristic of Arctic populations, implying their role in cold adaptation, we demonstrate that the surplus of nonsynonymous mutations is a general feature of the young branches of the phylogenetic tree, affecting also those that are found only in Africa. We introduce a new calibration method of the mutation rate of synonymous transitions to estimate the coalescent times of mtDNA haplogroups.

Background In the current context of tailoring interventions to maximize impact, it is important that current data of clinical epidemiology guide public health programmes and health workers in the management of severe disease. This study aimed at describing the burden of severe malaria at hospital level in two areas with distinct malaria transmission intensity. Methods A hospital-based surveillance was established in two regional hospitals located in two areas exposed to different malaria transmission. Data on paediatric severe malaria admissions were recorded using standardized methods from August 2017 to August 2018 with an interruption during the dry season from April to June 2018. Results In total, 921 children with severe malaria cases were enrolled in the study. The mean age was 33.9 (± 1.3) and 36.8 (± 1.6) months in lower malaria transmission (LMT) and higher malaria transmission (HMT) areas (p = 0.15), respectively. The geometric mean of asexual P. falciparum density was significantly higher in the LMT area compared to the HMT area: 22,861 trophozoites/µL (95% CI 17,009.2–30,726.8) vs 11,291.9 trophozoites/µL (95% CI 8577.9–14,864.5). Among enrolled cases, coma was present in 70 (9.2%) participants. 196 patients (21.8%) presented with two or more convulsions episodes prior to admission. Severe anaemia was present in 448 children (49.2%). Other clinical features recorded included 184 (19.9%) cases of lethargy, 99 (10.7%) children with incoercible vomiting, 80 (8.9%) patients with haemoglobinuria, 43 (4.8%) children with severe hypoglycaemia, 37 (4.0%) cases where child was unable to drink/suck, 11 (1.2%) cases of patients with circulatory collapse/shock, and 8 cases (0.9%) of abnormal bleeding (epistaxis). The adjusted odds of presenting with coma, respiratory distress, haemoglobinuria, circulatory collapse/shock and hypoglycaemia were significantly higher (respectively 6.5 (95%CI 3.4–12.1); 1.8 (95%CI 1.0–3.2); 2.7 (95%CI 1.6–4.3); 5.9 (95%CI 1.3–27.9); 1.9 (95%CI 1.0–3.6)) in children living in the HMT area compared to those residing in the LMT area. Overall, forty-four children died during hospitalization (case fatality rate 5.0%) with the highest fatalities in children admitted with respiratory distress (26.0%) and those with hypoglycaemia (25.0%). Conclusion The study showed that children in the HMT area have a higher risk of presenting with coma, shock/dehydration, haemoglobinuria, hypoglycaemia, and respiratory distress. Case-fatality rate is higher among patients with respiratory distress or hypoglycaemia. Hospital surveillance provides a reliable and sustainable means to monitor the clinical presentation of severe malaria and tailor the training needs and resources allocation for case management.

Background The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites. Aim and objectives The aim of this work was to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on detection and amplification of gametocyte sex specific transcripts selected from the literature: the female-specific pfs25 and pf glycerol kinase ( pfGK ) and the male-specific pfs230p and pf13 transcripts. Methods RTqPCR assays were used to test the gametocyte- and sex-specific expression of the target genes using asexual stages of the gametocyteless parasite clone F12 and FACS purified male and female gametocytes of the PfDynGFP/P47mCherry line. Assays were performed on 50 blood samples collected during an epidemiological survey in the Soumousso village, Burkina Faso, West-Africa, and amplification of the human housekeeping gene 18S rRNA was employed to normalize RNA sample variability. Results SYBR Green assays were developed that showed higher sensitivity compared to Taqman assays at a reduced cost. RTqPCR results confirmed that expression of pfs25 and pfs230p are female and male-specific, respectively, and introduced two novel markers, the female-specific pfGK and the male-specific pf13 . A formula was derived to calculate the ratio of male to female gametocytes based on the ratio of male to female transcript copy number. Use of these assays in the field samples showed, as expected, a higher sensitivity of RTqPCR compared to microscopy. Importantly, similar values of gametocyte sex-ratio were obtained in the field samples based on the four different target combinations. Conclusion Novel, sensitive, cheap and robust molecular assays were developed for the detection and quantification of female and male P. falciparum gametocytes. In particular, the RTqPCR assays based on the female-specific pfs25 and the newly described male gametocyte-specific pf13 transcripts, including normalization by the human 18S, reliably assess presence and abundance of female and male gametocytes and enable to determine their sex-ratio in human subjects in endemic areas.

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.

Assessment of exposure to malaria vectors is important to our understanding of spatial and temporal variations in disease transmission and facilitates the targeting and evaluation of control efforts. Recently, an immunogenic Anopheles gambiae salivary protein (gSG6) was identified and proposed as the basis of an immuno-assay determining exposure to Afrotropical malaria vectors. In the present study, IgG responses to gSG6 and 6 malaria antigens (CSP, AMA-1, MSP-1, MSP-3, GLURP R1, and GLURP R2) were compared to Anopheles exposure and malaria incidence in a cohort of children from Korogwe district, Tanzania, an area of moderate and heterogeneous malaria transmission. Anti-gSG6 responses above the threshold for seropositivity were detected in 15% (96/636) of the children, and were positively associated with geographical variations in Anopheles exposure (OR 1.25, CI 1.01-1.54, p = 0.04). Additionally, IgG responses to gSG6 in individual children showed a strong positive association with household level mosquito exposure. IgG levels for all antigens except AMA-1 were associated with the frequency of malaria episodes following sampling. gSG6 seropositivity was strongly positively associated with subsequent malaria incidence (test for trend p = 0.004), comparable to malaria antigens MSP-1 and GLURP R2. Our results show that the gSG6 assay is sensitive to micro-epidemiological variations in exposure to Anopheles mosquitoes, and provides a correlate of malaria risk that is unrelated to immune protection. While the technique requires further evaluation in a range of malaria endemic settings, our findings suggest that the gSG6 assay may have a role in the evaluation and planning of targeted and preventative anti-malaria interventions.

Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of \"natural\" parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ~40-fold coverage of the genome was observed per lane for samples with ≤ 30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ~40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum.

Background The Anopheles gambiae gSG6 is an anopheline-specific salivary protein which helps female mosquitoes to efficiently feed on blood. Besides its role in haematophagy, gSG6 is immunogenic and elicits in exposed individuals an IgG response, which may be used as indicator of exposure to the main African malaria vector A. gambiae . However, malaria transmission in tropical Africa is sustained by three main vectors ( A. gambiae , Anopheles arabiensis and Anopheles funestus ) and a general marker, reflecting exposure to at least these three species, would be especially valuable. The SG6 protein is highly conserved within the A. gambiae species complex whereas the A. funestus homologue, fSG6, is more divergent (80% identity with gSG6). The aim of this study was to evaluate cross-reactivity of human sera to gSG6 and fSG6. Methods The A. funestus SG6 protein was expressed/purified and the humoral response to gSG6, fSG6 and a combination of the two antigens was compared in a population from a malaria hyperendemic area of Burkina Faso where both vectors were present, although with a large A. gambiae prevalence (>75%). Sera collected at the beginning and at the end of the high transmission/rainy season, as well as during the following low transmission/dry season, were analysed. Results According to previous observations, both anti-SG6 IgG level and prevalence decreased during the low transmission/dry season and showed a typical age-dependent pattern. No significant difference in the response to the two antigens was found, although their combined use yielded in most cases higher IgG level. Conclusions Comparative analysis of gSG6 and fSG6 immunogenicity to humans suggests the occurrence of a wide cross-reactivity, even though the two proteins carry species-specific epitopes. This study supports the use of gSG6 as reliable indicator of exposure to the three main African malaria vectors, a marker which may be useful to monitor malaria transmission and evaluate vector control measures, especially in conditions of low malaria transmission and/or reduced vector density. The Anopheles stephensi SG6 protein also shares 80% identity with gSG6, suggesting the attractive possibility that the A. gambiae protein may also be useful to assess human exposure to several Asian malaria vectors.

BACKGROUND: Mosquito saliva plays crucial roles in blood feeding but also evokes in hosts an anti-saliva antibody response. The IgG response to the Anopheles gambiae salivary protein gSG6 was previously shown to be a reliable indicator of human exposure to Afrotropical malaria vectors. We analyzed here the humoral response to the salivary anti-thrombin cE5 in a group of individuals from a malaria hyperendemic area of Burkina Faso. METHODS: ELISA was used to measure the anti-cE5 IgG, IgG1 and IgG4 antibody levels in plasma samples collected in the village of Barkoumbilen (Burkina Faso) among individuals of the Rimaibé ethnic group. Anti-gSG6 IgG levels were also determined for comparison. Anopheles vector density in the study area was evaluated by indoor pyrethrum spray catches. RESULTS: The cE5 protein was highly immunogenic and triggered in exposed individuals a relatively long-lasting antibody response, as shown by its unchanged persistence after a few months of absent or very low exposure (dry season). In addition cE5 did not induce immune tolerance, as previously suggested for the gSG6 antigen. Finally, IgG subclass analysis suggested that exposed individuals may mount a Th1-type immune response against the cE5 protein. CONCLUSIONS: The anti-cE5 IgG response is shown here to be a sensitive indicator of human exposure to anopheline vectors and to represent an additional tool for malaria epidemiological studies. It may be especially useful in conditions of low vector density, to monitor transiently exposed individuals (i.e. travellers/workers/soldiers spending a few months in tropical Africa) and to evaluate the impact of insecticide treated nets on vector control. Moreover, the gSG6 and cE5 salivary proteins were shown to trigger in exposed individuals a strikingly different immune response with (i) gSG6 evoking a short-lived IgG response, characterized by high IgG4 levels and most likely induction of immune tolerance, and (ii) cE5 eliciting a longer-living IgG response, dominated by anti-cE5 IgG1 antibodies and not inducing tolerance mechanisms. We believe that these two antigens may represent useful reagents to further investigate the so far overlooked role of Anopheles saliva and salivary proteins in host early immune response to Plasmodium parasites.

Human antibody response to the Anopheles gambiae salivary protein gSG6 has recently emerged as a potentially useful tool for malaria epidemiological studies and for the evaluation of vector control interventions. However, the current understanding of the host immune response to mosquito salivary proteins and of the possible crosstalk with early response to Plasmodium parasites is still very limited. We report here the analysis of IgG1 and IgG4 subclasses among anti-gSG6 IgG responders belonging to Mossi and Fulani from Burkina Faso, two ethnic groups which are known for their differential humoral response to parasite antigens and for their different susceptibility to malaria. The IgG1 antibody response against the gSG6 protein was comparable in the two groups. On the contrary, IgG4 titers were significantly higher in the Fulani where, in addition, anti-gSG6 IgG4 antibodies appeared in younger children and the ratio IgG4/IgG1 stayed relatively stable throughout adulthood. Both gSG6-specific IgG1 and IgG4 antibodies showed a tendency to decrease with age whereas, as expected, the IgG response to the Plasmodium circumsporozoite protein (CSP) exhibited an opposite trend in the same individuals. These observations are in line with the idea that the An. gambiae gSG6 salivary protein induces immune tolerance, especially after intense and prolonged exposure as is the case for the area under study, suggesting that gSG6 may trigger in exposed individuals a Th2-oriented immune response.