Absence of the human CYP2C8*3 allele in Ugandan children exposed to Plasmodium falciparum malaria.

Study of host pharmacogenetics can improve our knowledge of mechanisms of drug resistance selection and spread. This issue has recently been addressed with respect to chloroquine and amodiaquine in malaria endemic areas of West and East Africa. Here we report, from surveys performed in two different areas of Uganda, that the human CYP2C8*3 allele, which had been reported to be strongly associated with parasite drug resistance in Zanzibar, is absent, being a marker of genetic admixture of the Zanzibari population with a Caucasoid component. Moreover, a retrospective analysis of CYP2C8*2 and the Plasmodium falciparum drug resistant pfmdr1 86Y allele does not show any association, which may be related to the high level of drug resistance.

Looking for the gold standard: assessment of the effectiveness of four traps for monitoring mosquitoes in Italy.

Several kinds of traps are available for the collection of Culicidae species creating nuisance problems and/or a potential risk of pathogen transmission. The choice of the most appropriate sampling device should take into consideration the objective of the monitoring activity (e.g., faunistic research, vector control evaluation, arbovirus surveillance, etc.), the ecological and behavioral characteristics of the target mosquito species, and the ecology of the sampling areas. However, there are few factual criteria technical personnel can rely on to choose the most suitable sampling method, particularly when the targets are represented by mosquito species in temperate areas. We carried out a Latin square experiment in three ecologically different settings in Mantua municipality (northern Italy) to compare the performance of four different traps targeting host-seeking mosquitoes: two traps specifically designed for mosquito monitoring purposes (Centers for Disease Control and Prevention CO(2) trap and Biogents BG Eisenhans de Luxe trap) and two designed to reduce mosquito densities in outdoor domestic settings (Activa Acti Power Trap PV 440 and Activa Acti Power Trap MT 250 Plus). Overall, 1,930 specimens belonging to nine species were collected and differences in the performance of the four traps with reference to their ability to detect overall species diversity, as well as to collect single species, were highlighted. These observations, coupled with an analysis of the costs associated with the trap's purchase, operation, and servicing, provide useful indications for the implementation of mosquito monitoring in temperate areas.

Distribution of human CYP2C8*2 allele in three different African populations.

BACKGROUND: The aim of this study was to investigate cytochrome P450 2C8*2 (CYP2C8*2) distribution and allele frequency in three populations from West and East Africa exposed to Plasmodium falciparum malaria. CYP2C8 enzyme is involved in the metabolism of the anti-malarials amodiaquine and chloroquine. The presence of the CYP2C8*2 defective allele has been recently associated to higher rate of chloroquine-resistant malaria parasites. METHODS: A total of 503 young subjects were genotyped for the single nucleotide polymorphism rs11572103 (A/T). Eighty-eight were from southern Senegal, 262 from eastern Uganda and 153 from southern Madagascar. The PCR-RFLP technique was used to discriminate the wild-type (A) from the defective allele (T). RESULTS: A CYP2C8*2 (T) allele frequency of 0.222 ± 0.044 was detected in Senegal, 0.105 ± 0.019 in Uganda and 0.150 ± 0.029 in Madagascar. CONCLUSIONS: This study demonstrated that CYP2C8*2 allele is widespread in Africa. This allele occurs at different frequency in West and East Africa, being higher in Senegal than in Uganda and Madagascar. These data indicate that an important fraction of the populations analysed has a decreased enzymatic activity, thus being at higher risk for drug accumulation with two possible consequences: i) an exacerbation of drug-associated adverse side effects; ii) an increase of drug-resistance selection pressure on P. falciparum parasites.

Human genetic variation is associated with Plasmodium falciparum drug resistance.

One approach to investigate if human genetic variation influences the selection of Plasmodium falciparum drug resistance is to compare the frequency of resistant infections among human populations differing in their genetic background and living in the same epidemiological context. A further complementary approach consists in comparing drug resistance among subjects differing for genes involved in drug metabolism. Here we report, from malariological surveys performed in Burkina Faso, that the prevalence of P. falciparum chloroquine-resistant infections (pfcrt 76T and/or pfmdr1 86Y alleles) differs among sympatric ethnic groups, being higher in the Mossi and Rimaibé groups than in the Fulani group (odds ratio [OR], 2.24; 95% confidence interval [CI], 1.27-3.92; P = .007). The association analysis revealed that the human CYP2C8*2 variant, known to determine a poor drug metabolizer phenotype, was associated with P. falciparum chloroquine-resistant infections (OR, 1.66; 95% CI, 1.13-2.43; P = .008). This variant is more frequent in the Mossi-Rimaibé group (23.7% ± 1.4%) than in the Fulani group (9.9% ± 2.5%; P = .0003). This study provides an example of how host genetic variation may influence the selection dynamics of a pathogen's drug resistance.

Humoral response to the Anopheles gambiae salivary protein gSG6: a serological indicator of exposure to Afrotropical malaria vectors.

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.

The Anopheles gambiae salivary protein gSG6: an anopheline-specific protein with a blood-feeding role.

The Anopheles gambiae salivary gland protein 6 (gSG6) is a small protein specifically found in the salivary glands of adult female mosquitoes. We report here the expression of a recombinant form of the protein and we show that in vivo gSG6 is expressed in distal-lateral lobes and is secreted with the saliva while the female mosquito probes for feeding. Injection of gSG6 dsRNA into adult A. gambiae females results in decreased gSG6 protein levels, increased probing time and reduced blood feeding ability. gSG6 orthologs have been found so far only in the salivary glands of Anopheles stephensi and Anopheles funestus, both members of the Cellia subgenus. We report here the gSG6 sequence from five additional anophelines, four species of the A. gambiae complex and Anopheles freeborni, a member of the subgenus Anopheles. We conclude that gSG6 plays some essential blood feeding role and was recruited in the anopheline subfamily most probably after the separation of the lineage which gave origin to Cellia and Anopheles subgenera.

Analysis of apyrase 5' upstream region validates improved Anopheles gambiae transformation technique.

BACKGROUND: Genetic transformation of the malaria mosquito Anopheles gambiae has been successfully achieved in recent years, and represents a potentially powerful tool for researchers. Tissue-, stage- and sex-specific promoters are essential requirements to support the development of new applications for the transformation technique and potential malaria control strategies. During the Plasmodium lifecycle in the invertebrate host, four major mosquito cell types are involved in interactions with the parasite: hemocytes and fat body cells, which provide humoral and cellular components of the innate immune response, midgut and salivary glands representing the epithelial barriers traversed by the parasite during its lifecycle in the mosquito. FINDINGS: We have analyzed the upstream regulatory sequence of the An. gambiae salivary gland-specific apyrase (AgApy) gene in transgenic An. gambiae using a piggyBac transposable element vector marked by a 3xP3 promoter:DsRed gene fusion. Efficient germ-line transformation in An. gambiae mosquitoes was obtained and several integration events in at least three different G0 families were detected. LacZ reporter gene expression was analyzed in three transgenic lines/groups, and in only one group was tissue-specific expression restricted to salivary glands. CONCLUSION: Our data describe an efficient genetic transformation of An. gambiae embryos. However, expression from the selected region of the AgApy promoter is weak and position effects may mask tissue- and stage- specific activity in transgenic mosquitoes.

Chromosomal plasticity and evolutionary potential in the malaria vector Anopheles gambiae sensu stricto: insights from three decades of rare paracentric inversions.

BACKGROUND: In the Anopheles gambiae complex, paracentric chromosomal inversions are non-randomly distributed along the complement: 18/31 (58%) of common polymorphic inversions are on chromosome arm 2R, which represents only approximately 30% of the complement. Moreover, in An. gambiae sensu stricto, 6/7 common polymorphic inversions occur on 2R. Most of these inversions are considered markers of ecological adaptation that increase the fitness of the carriers of alternative karyotypes in contrasting habitats. However, little is known about the evolutionary forces responsible for their origin and subsequent establishment in field populations. RESULTS: Here, we present data on 82 previously undescribed rare chromosomal inversions (RCIs) recorded during extensive field sampling in 16 African countries over a 30 year period, which may shed light on the dynamics of chromosomal plasticity in An. gambiae. We analyzed breakpoint distribution, length, and geographic distribution of RCIs, and compared these measures to those of the common inversions. We found that RCIs, like common inversions, are disproportionately clustered on 2R, which may indicate that this arm is especially prone to breakages. However, contrasting patterns were observed between the geographic distribution of common inversions and RCIs. RCIs were equally frequent across biomes and on both sides of the Great Rift Valley (GRV), whereas common inversions predominated in arid ecological settings and west of the GRV. Moreover, the distribution of RCI lengths followed a random pattern while common inversions were significantly less frequent at shorter lengths. CONCLUSION: Because 17/82 (21%) RCIs were found repeatedly at very low frequencies - at the same sampling location in different years and/or in different sampling locations - we suggest that RCIs are subject mainly to drift under unperturbed ecological conditions. Nevertheless, RCIs may represent an important reservoir of genetic variation for An. gambiae in response to environmental changes, further testifying to the considerable evolutionary potential hidden within this pan-African malaria vector.

Exploring proteins in Anopheles gambiae male and female antennae through MALDI mass spectrometry profiling.

MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.

Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria.

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNgamma, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+ CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFbeta, TGFbetaRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFbeta and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

High human herpesvirus 8 seroprevalence in populations from Western Balkan countries.

Patterns of endemicity of human herpesvirus 8 (HHV8) are still undefined in some European populations, such as those from Western Balkan countries. Serum samples from 605 human immunodeficiency virus-seronegative subjects (299 Albanians and 306 Kosovars) were tested for the presence of HHV8 antibodies to a capsid-related open reading frame (ORF65)-encoded protein and a latency-associated nuclear antigen (LANA) to determine HHV8 seroprevalence in populations from Albania and from the Kosovo region of former Yugoslavia. Levels of co- circulation with hepatitis A (HAV) and hepatitis B (HBV) viruses were also determined. HHV8 antibodies to at least one of the two antigens were detected in 28.8% of Albanians and 18% of Kosovars. The seroprevalence of HHV8 was found to be 25.0 and 16.8% in Albanian and Kosovar children (

Identification and composition of cuticular hydrocarbons of the major Afrotropical malaria vector Anopheles gambiae s.s. (Diptera: Culicidae): analysis of sexual dimorphism and age-related changes.

Forty-eight cuticular hydrocarbons (CHCs) were characterized by gas chromatography-mass spectrometry from the epicuticular surface of the major Afrotropical malaria vector Anopheles gambiae. The hydrocarbons identified were 14 n-alkanes, 16 monomethyl alkanes, 13 dimethyl alkanes, 5 alkenes, with main-chain lengths ranging from C(17) to C(47), and the results are consistent with those from other Culicidae species. Qualitative differences were not observed between laboratory pools of three females and males, between different age-groups (0-16 days) and between single field specimens, whereas quantitative differences in CHC profiles were observed. Differences between sexes were more marked in individuals aged 0-2 days than in older ones. Both sexes undergo strong CHC profile changes with age, and individuals aged 0-2 days differ remarkably from the older ones. The possibility of exploiting these changes for estimating the age of mosquito was explored through multivariate analyses of the relative abundance of the compounds, using either the whole CHC profile or a subset of CHCs. Such a method allows us to assign more than 85% of females and 75% of males to the correct age-group. Although preliminary, these results show that the method is promising, as it has already been shown in Aedes aegypti and An. stephensi. The correct determination of the vector age (particularly in the case of the An. gambiae complex of sibling species) provides valuable information in malaria epidemiology and in evaluation of the effectiveness of vector control strategies. Further efforts will be made to validate this method on single specimens reared in seminatural conditions before being proposed to medical entomologists working in the Afrotropical region.

An updated catalogue of salivary gland transcripts in the adult female mosquito, Anopheles gambiae.

Salivary glands of blood-sucking arthropods contain a variety of compounds that prevent platelet and clotting functions and modify inflammatory and immunological reactions in the vertebrate host. In mosquitoes, only the adult female takes blood meals, while both sexes take sugar meals. With the recent description of the Anopheles gambiae genome, and with a set of approximately 3000 expressed sequence tags from a salivary gland cDNA library from adult female mosquitoes, we attempted a comprehensive description of the salivary transcriptome of this most important vector of malaria transmission. In addition to many transcripts associated with housekeeping functions, we found an active transposable element, a set of Wolbachia-like proteins, several transcription factors, including Forkhead, Hairy and doublesex, extracellular matrix components and 71 genes coding for putative secreted proteins. Fourteen of these 71 proteins had matching Edman degradation sequences obtained from SDS-PAGE experiments. Overall, 33 transcripts are reported for the first time as coding for salivary proteins. The tissue and sex specificity of these protein-coding transcripts were analyzed by RT-PCR and microarray experiments for insight into their possible function. Notably, two gene products appeared to be differentially spliced in the adult female salivary glands, whereas 13 contigs matched predicted intronic regions and may include additional alternatively spliced transcripts. Most An. gambiae salivary proteins represent novel protein families of unknown function, potentially coding for pharmacologically or microbiologically active substances. Supplemental data to this work can be found at http://www.ncbi.nlm.nih.gov/projects/omes/index.html#Ag2.

Chromosome speciation: humans, Drosophila, and mosquitoes.

Chromosome rearrangements (such as inversions, fusions, and fissions) may play significant roles in the speciation between parapatric (contiguous) or partly sympatric (geographically overlapping) populations. According to the "hybrid-dysfunction" model, speciation occurs because hybrids with heterozygous chromosome rearrangements produce dysfunctional gametes and thus have low reproductive fitness. Natural selection will, therefore, promote mutations that reduce the probability of intercrossing between populations carrying different rearrangements and thus promote their reproductive isolation. This model encounters a disabling difficulty: namely, how to account for the spread in a population of a chromosome rearrangement after it first arises as a mutation in a single individual. The "suppressed-recombination" model of speciation points out that chromosome rearrangements act as a genetic filter between populations. Mutations associated with the rearranged chromosomes cannot flow from one to another population, whereas genetic exchange will freely occur between colinear chromosomes. Mutations adaptive to local conditions will, therefore, accumulate differentially in the protected chromosome regions so that parapatric or partially sympatric populations will genetically differentiate, eventually evolving into different species. The speciation model of suppressed recombination has recently been tested by gene and DNA sequence comparisons between humans and chimpanzees, between Drosophila species, and between species related to Anopheles gambiae, the vector of malignant malaria in Africa.

Difference in susceptibility to malaria between two sympatric ethnic groups in Mali.

We compared malaria indicators among sympatric groups to study human heterogeneities in the response to Plasmodium falciparum malaria infection. Four cross-sectional surveys and two longitudinal surveys in two sympatric ethnic groups (Dogon and Fulani) in Mali were carried out from 1998 to 2000. Spleen and parasite rates were evaluated during the cross-sectional surveys and disease incidence was assessed during longitudinal surveys. In spite of similar sociocultural factors and entomologic inoculation rates between ethnic groups, the Fulani had a significantly higher spleen enlargement rate, lower parasite rate, and were less affected by the disease than the Dogon group, whose frequency of hemoglobin C was higher than that recorded among the Fulani group. The Fulani group had significantly higher levels of IgG and IgE against crude malaria antigen than the Dogon group, suggesting a role of anti-malaria antibodies in the immune protection seen in this group.

Lessons learned from malaria: Italy's past and sub-Sahara's future.

No longer a major public health concern in developed countries, malaria kills 1-3 million people annually, mostly children under the age of five in sub-Saharan Africa. In 1998, the WHO launched the Roll Back Malaria (RBM) drive to halve malaria mortality by 2010. This article contrasts the problems confronting RBM with the successful Italian drive to eradicate malaria between the late 19th and mid 20th centuries. The Italians employed education and applied socio-political will; however, ecological and socio-economic conditions in sub-Saharan Africa are more hospitable to the disease. RBM strategies should consider the Italian experience while awaiting a major scientific breakthrough necessary to achieve success.