Comparison of the Performances of Five Primer Sets for the Detection and Quantification of Plasmodium in Anopheline Vectors by Real-Time PCR.

Quantitative real-time polymerase chain reaction (qrtPCR) has made a significant improvement for the detection of Plasmodium in anopheline vectors. A wide variety of primers has been used in different assays, mostly adapted from molecular diagnosis of malaria in human. However, such an adaptation can impact the sensitivity of the PCR. Therefore we compared the sensitivity of five primer sets with different molecular targets on blood stages, sporozoites and oocysts standards of Plasmodium falciparum (Pf) and P. vivax (Pv). Dilution series of standard DNA were used to discriminate between methods at low concentrations of parasite and to generate standard curves suitable for the absolute quantification of Plasmodium sporozoites. Our results showed that the best primers to detect blood stages were not necessarily the best ones to detect sporozoites. Absolute detection threshold of our qrtPCR assay varied between 3.6 and 360 Pv sporozoites and between 6 and 600 Pf sporozoites per mosquito according to the primer set used in the reaction mix. In this paper, we discuss the general performance of each primer set and highlight the need to use efficient detection methods for transmission studies.

A multiplex PCR to differentiate the two sibling species of mosquitoes Ochlerotatus detritus and Oc. coluzzii and evidence for further genetic heterogeneity within the Detritus complex.

Internal transcribed spacer regions of ribosomal DNA were sequenced, and species-specific primers were designed to simplify the identification of two morphologically similar species of the Detritus complex, Ochlerotatus detritus and Ochlerotatuscoluzzii. Each newly designed primer was able to amplify a species-specific fragment with a different size. Samples from France and Germany were successfully tested. This new tool prompts for bio-ecological studies to refine basic knowledge on the bionomics of this species complex, towards a better control and prevention of ensuing nuisances. Moreover, ITS2 sequencing revealed the existence of (1) two distinct haplotypes of Oc. detritus that are sympatric and widely distributed along the French Atlantic and Mediterranean littorals and (2) a specific haplotype in mosquitoes sampled from Tunisia, raising the question of the taxonomic status of this North-African population.

Molecular and morphological systematics of the sandfly Sergentomyia (Sintonius) clydei Sinton, 1928 and questions about its record in the Seychelles.

In the Phlebotomine sandflies, a few molecular studies related on the genus Sergentomyia have been published. The present study explored the genetic variability within Sergentomyia (Sintonius) clydei (Diptera, Psychodidae). The sampling included 15 populations originating from 12 countries. A morphological approach was coupled to the sequencing of two molecular markers (cytochrome b mtDNA and cacophony nuclear DNA). The most variable morphological characters resided in the cibarium of the females, especially (i) the pigment patch pattern and (ii) the number of cibarial teeth and denticles in the armature. However this morphological approach was unable to individualize any population within S. clydei. The NJ trees based on both molecular markers individualized the specimens from the Aldabra group of islands in the Seychelles. Surprisingly, cyt b variability was not compatible with the known data about the complete submersion of Aldabra occurring relatively recently some 125,000 years ago. The settlement of these islands by S. clydei from continental Africa, the Middle East or Asia, and the value of mtDNA markers are discussed.

[Updated inventory of mosquitoes (Diptera: Culicidae) of the island of La Réunion, Indian Ocean]. / Inventaire actualisé des moustiques (Diptera : Culicidae) de l'île de La Réunion, océan Indien.

A literature analysis coupled with new entomological surveys conducted between 2009 and 2012 led to changes in the list of mosquito species present on the island of La Réunion. Using morphological criteria, Orthopodomyia arboricollis is replaced by Or. reunionensis. On the basis of morphometrical and genetic criteria, Culex univittatus is replaced by Cx. neavei. Cx. poicilipes, which was already reported missing 40 years ago, has not been found again. Anopheles arabiensis is confirmed as the only species of the Gambiae complex present on the island. Thus, twelve species are currently known. For each of them, elements of taxonomic, biological and medical interest are listed. An. arabiensis is a major vector of human Plasmodium (last case of indigenous malaria in 1967). In the Indian Ocean, Aedes albopictus and Ae. aegypti both are competent for transmitting dengue and chikungunya viruses. In Africa, Cx. quinquefasciatus transmits Wuchereria bancrofti and Cx. neavei transmits the Sindbis virus; both species also transmit the West Nile virus. Cx. tritaeniorhynchus is the major vector of Japanese Encephalitis virus in Asia. Two species are endemic (Ae. dufouri and Or. reunionensis), the ten other ones are also found in Madagascar and on the African continent (An. coustani, An. arabiensis, Ae. fowleri, Cx. tritaeniorhynchus, Cx. neavei, Cx. insignis, Lutzia tigripes), with three of them having also a cosmopolitan distribution (Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus). Among the twelve recorded taxa, eight species are anthropophilic, three are supposedly zoophilic and one is a predatory species. No new invasive anthropophilic species did settle on the island. Updated identification keys of larval and adult stages are proposed.

First record of Phlebotomine sandflies (Diptera: Psychodidae) in the Comoros Archipelago with description of Sergentomyia (Vattieromyia) pessoni n. sp. and S. (Rondanomyia) goodmani comorensis n. ssp.

No Phlebotomine sandflies had ever been reported in the Comoros Archipelago, including the three islands of the Republic of the Union of Comoros (Grande Comore, Mohéli and Anjouan) and the French oversea department of Mayotte. During three field surveys carried out in 2003, 2007 and 2011, we provided the first record of Phlebotomine sandflies in this area. A total of 85 specimens belonging to three species were caught: a new species S. (Vattieromyia) pessoni n. sp. (two females from Grande Comore), a new subspecies of Sergentomyia (Rondanomyia) goodmani (80 specimens from Grande Comore and one from Anjouan) and Grassomyia sp. (two females from Mohéli). The individualisation of these taxa was inferred both from morphological criteria and sequencing of a part of the cytochrome b of the mitochondrial DNA. These taxa are closely related to Malagasy sandflies.

Physiology and development of the M and S molecular forms of Anopheles gambiae in Burkina Faso (West Africa).

In West Africa, M and S molecular forms of Anopheles gambiae sensu stricto (Diptera: Culicidae) Giles, frequently occur together, although with different population bionomics. The S form typically breeds in rain-dependant water collections and is present during the rainy season only whereas the M form can thrive all year long in areas with permanent breeding opportunities. In the present study, we explored physiological and developmental trade-offs at play in laboratory colonies and field populations of the M and S forms that originated from an area of sympatry in Burkina Faso, where M and S larvae exhibit such habitat segregation. In the laboratory, larvae of the M form developed slower than the S form (mean values 9.51 and 8.85 days, respectively, Wilcoxon's test, P < 0.001). Although wing length and dry weight at emergence showed large variations, M females were on average 8% heavier than S females of similar wing length. Higher nutritional reserves (proteins and lipids) in teneral adults explained part of this weight difference, reflecting a better ability of the M form to garner resources at the larval stage. Furthermore, a higher rate of ovarian maturation was observed in the M form after a single bloodmeal. The relevance of these findings for parasite transmission is discussed.

[Resistance of Anopheles labranchiae to DDT in Morocco: identification of the mechanisms and choice of replacement insecticide]. / Résistance d'Anopheles labranchiae au DDT au Maroc: identification des mécanismes et choix d'un insecticide de remplacement.

A study of Anopheles labranchiae resistance in Morocco was conducted in the provinces of Kénitra, Khouribga, Larache, Khémisset and Salé during 2005. An. labranchiae was susceptible to propoxur, fenitrothion and permethrin and resistant to varying degrees to DDT. Genetically there was no change to the target site common to DDT and pyrethroids, the voltage gated sodium channel. The resistance seemed to be due to detoxification mechanisms specific to DDT. In principle, there should be no obstacle to the substitution of DDT by pyrethroids in Morocco. Resistance can then be detected and supervised by more reliable molecular tools in the Laboratory of Medical Entomology of the National Institute of Hygiene.

[Resistance of Anopheles labranchiae to DDT in Morocco: identification of the mechanisms and choice of replacement insecticide]

A study of Anopheles labranchiae resistance in Morocco was conducted in the provinces of K‚nitra, Khouribga, Larache, Kh‚misset and Sal‚ during 2005. An. labranchiae was susceptible to propoxur, fenitrothion and permethrin and resistant to varying degrees to DDT. Genetically there was no change to the target site common to DDT and pyrethroids, the voltage gated sodium channel. The resistance seemed to be due to detoxification mechanisms specific to DDT. In principle, there should be no obstacle to the substitution of DDT by pyrethroids in Morocco. Resistance can then be detected and supervised by more reliable molecular tools in the Laboratory of Medical Entomology of the National Institute of Hygiene

Population dynamics of pest mosquitoes and potential malaria and West Nile virus vectors in relation to climatic factors and human activities in the Camargue, France.

The Camargue is an extensive wetland in the southeast of France, which is highly influenced by human activities. Large ponds, marshes and irrigated fields provide abundant potential breeding sites for mosquitoes. mosquitoes, which are important in terms of the nuisance they cause to people and animals, the limitations they impose on tourism and their potential threat to human health. Several of the mosquito species present are potential vectors of malaria and West Nile virus. Therefore, the population dynamics of these species were monitored over an entire breeding season during March-October 2005. Mosquito populations were sampled in two study areas once every 2 weeks, using CDC light traps baited with CO(2). Sixteen species were collected. The majority (98.7%) of the catch were Aedes caspius (Pallas) (Diptera: Culicidae), Culex modestus (Ficalbi), Culex pipiens L. and Anopheles hyrcanus (Pallas). The population dynamics of these species varied considerably in relation to the species' biology, climatic conditions (rainfall, temperature and season), water management, implementation of mosquito control campaigns and landscape use.

Multiple insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus from Benin, West Africa.

Because free-insecticide treated net distribution is planned in Benin (West Africa) during the next few years, we investigated the type, frequency and distribution of insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus mosquitoes in four localities selected on the basis of contrasting agricultural practices, use of insecticides and environment. Bioassays with WHO diagnostic test kits were carried out using pyrethroid, carbamate, organophosphate and organochlorine insecticides. An. gambiae mosquitoes were identified to species and to M or S molecular forms using PCR techniques. Molecular and biochemical assays were carried out to identify kdr and Ace.1 mutations in individual mosquitoes and to detect any increase in the activity of enzymes typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transférases). WHO diagnostic tests showed high frequency of resistance in An. gambiae and Cx. quinquefasciatus to permethrin and DDT in three areas. This was consistent with the presence of target site insensitivity due to kdr mutation and to increased metabolism through enzymatic activity. Kdr was expressed in both M and S forms. However, less than 1% of An. gambiae or Cx. quiqnuefasciatus showed the presence of the Ace.1(R) mutation. Carbamate/OP resistance was present at higher frequency in Culex than in An. gambiae. Dieldrin resistance was present in both species at all four localities. A higher frequency of pyrethroid-resistance was found in An. gambiae mosquitoes collected in urban areas compared to those collected in rice growing areas. The expansion of vegetable growing within urban areas probably contributed to selection pressure on mosquitoes. The detection of multiple resistance mechanisms in both An. gambiae and Cx. quinquefasciatus in Benin may represent a threat for the efficacy of ITNs and other forms of vector control such as indoor residual spraying in the future.

The spread of the Leu-Phe kdr mutation through Anopheles gambiae complex in Burkina Faso: genetic introgression and de novo phenomena.

During extensive sampling in Burkina Faso and other African countries, the Leu-Phe mutation producing the kdr pyrethroid resistance phenotype was reported in both Anopheles gambiae ss and A. arabiensis. This mutation was widely distributed at high frequency in the molecular S form of A. gambiae while it has been observed at a very low frequency in both the molecular M form and A. arabiensis in Burkina Faso. While the mutation in the M form is inherited through an introgression from the S form, its occurrence is a new and independent mutation event in A. arabiensis. Three nucleotides in the upstream intron of the kdr mutation differentiated A. arabiensis from A. gambiae ss and these specific nucleotides were associated with kdr mutation in A. arabiensis. Ecological divergences which facilitated the spread of the kdr mutation within the complex of A. gambiae ss in West Africa, are discussed.

Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene.

Samples of the dengue vector mosquito Aedes aegypti (L.) (Diptera: Culicidae) were collected from 13 localities between 1995 and 1998. Two laboratory strains, Bora (French Polynesia) and AEAE, were both susceptible to DDT and permethrin; all other strains, except Larentuka (Indonesia) and Bouaké (Ivory Coast), contained individual fourth-instar larvae resistant to permethrin. Ten strains were subjected to a range of biochemical assays. Many strains had elevated carboxylesterase activity compared to the Bora strain; this was particularly high in the Indonesian strains Salatiga and Semarang, and in the Guyane strain (Cayenne). Monooxygenase levels were increased in the Salatiga and Paea (Polynesia) strains, and reduced in the two Thai strains (Mae Kaza, Mae Kud) and the Larentuka strain. Glutathione S-transferase activity was elevated in the Guyane strain. All other enzyme profiles were similar to the susceptible strain. The presence of both DDT and pyrethroid resistance in the Semarang, Belem (Brazil) and Long Hoa (Vietnam) strains suggested the presence of a knock-down resistant (kdr)-type resistance mechanism. Part of the S6 hydrophobic segment of domain II of the voltage-gated sodium channel gene was obtained by RT-PCR and sequenced from several insects from all 13 field strains. Four novel mutations were identified. Three strains contained identical amino acid substitutions at two positions, two strains shared a different substitution, and one strain was homozygous for a fourth alteration. The leucine to phenylalanine substitution that confers nerve insensitivity to pyrethroids in a range of other resistant insects was absent. Direct neurophysiological assays on individual larvae from three strains with these mutations demonstrated reduced nerve sensitivity to permethrin or lambda cyhalothrin inhibition compared to the susceptible strains.

Natural swarming behaviour of the molecular M form of Anopheles gambiae.

In Anopheles gambiae, as in most species of mosquitoes, mating is initiated in flight. The males aggregate in aerial swarms and conspecific females individually fly to these swarms where they mate with males. In this study, we investigated the swarming behaviour of A. gambiae and conducted 2 surveys in the rice field area of the Vallée du Kou in Burkina Faso in 1999 and 2002. A high number of anopheline mosquitoes were observed in this area and both molecular M and S forms of A. gambiae were found in sympatry. Swarms formed a few minutes after sunset in different places and no obvious markers were associated with their occurrence. However, swarms occurred close to cow herds generally in open flat areas, 2-3 m above the ground. Overall, 2829 anopheline mosquitoes were collected from 21 swarms composed primarily of males. A few specimens of Culex quinquefasciatus were collected from 3 swarms. Although both molecular M and S forms were found in sympatry in the village, swarms were composed almost exclusively of the molecular M form. This suggests that there are alternative swarming habits for both molecular M and S forms of A. gambiae in nature.

First report of the kdr mutation in Anopheles gambiae M form from Burkina Faso, west Africa.

The kdr mutation, conferring resistance to pyrethroid insecticides, has been reported in several West-African populations of Anopheles gambiae S form and in the M form populations from tropical forest of Benin. We report the finding of a single M specimen collected in the rice-field area of Vallée du Kou (Burkina Faso) showing the mutation at the heterozygous state. The monitoring of kdr mutation in An. gambiae forms/species is of paramount importance to implement effective malaria control tools and may greatly improve the knowledge of the relationship between and within An. gambiae populations.

The kdr mutation occurs in the Mopti form of Anopheles gambiae s.s. through introgression.

Anopheles gambiae s.s. is a complex of sibling taxa characterized by various paracentric inversions. In west and central Africa, where several taxa are sympatric, a kdr mutation responsible for pyrethroid resistance has been described in only one (the S taxon), suggesting an absence of gene flow between them. Following a thorough sampling, we have found a kdr mutation in another taxon (M). To establish whether this mutation is the same event or not, the large intron upstream of the kdr mutation was sequenced to find polymorphic sites in susceptible/resistant and M/S mosquitoes. The low genetic diversity found in this DNA region indicates that a local genetic sweep has recently occurred. However, some polymorphic sites were found, and it is therefore concluded that the kdr mutation in the M taxon is not an independent mutation event, and is best explained by an introgression from the S taxon. These results are discussed within the context of possible gene flow between members of An. gambiae s.s. taxa, and with the possible spread of the kdr mutation in other closely related malaria vectors of the An. gambiae complex.

Pyrethroid cross resistance spectrum among populations of Anopheles gambiae s.s. from Côte d'Ivoire.

Field samples of Anopheles gambiae s.s. from Côte d'Ivoire were tested with 5 pyrethroids (cyfluthrin, lambda-cyhalothrin, alpha-cypermethrin, deltamethrin, permethrin), 1 pseudo-pyrethroid (etofenprox), and an organochlorine (DDT). With the use of World Health Organization diagnostic tests, 5 out of 6 samples were found cross-resistant to these insecticides. A strong decrease in knockdown effect and mortality was also observed when testing deltamethrin-impregnated nettings. With a polymerase chain reaction amplification of specific alleles diagnostic test, resistance was found associated with the presence of a kdr mutation. The strong correlation between kdr allelic frequency and resistance to DDT or etofenprox indicated that kdr was the main resistance factor for these 2 insecticides. On the contrary, a lower correlation was observed between kdr frequency and resistance to 4 of the 5 pyrethroids tested, suggesting that another mechanism was also involved, likely a metabolic detoxification. These results point out the necessity to monitor pyrethroid resistance and the presence of kdr before implementation of any impregnated bed-net programs for malaria control.

Current distribution of a pyrethroid resistance gene (kdr) in Anopheles gambiae complex from west Africa and further evidence for reproductive isolation of the Mopti form.

In the field, the kdr mutation, involved in pyrethroid resistance, has been found widely distributed in the Savanna form of Anopheles gambiae s.s., but never in wild populations of the Mopti form or An. arabiensis, even in areas where both occur in sympatry with resistant Savanna populations. Under laboratory conditions, Mopti and Savanna forms were fully able to interbreed and the kdr mutation was transmissible from one form to the other. Both forms appeared to be exposed to pyrethroid selection pressure in the field. The absence of the kdr mutation in the Mopti form and the total lack of Mopti-Savanna heterozygotes in field populations provides further evidence of a pre-copulatory barrier to gene flow between these two forms. Molecular markers, including kdr, are powerful tools for studying population genetics and circulation of resistance genes, and should be used through an integrated approach for a better understanding of the speciation process.

HIV type 1 diversity and the reliability of the heteroduplex mobility assay.

We investigated HIV-1 diversity by means of heteroduplex mobility assay (HMA) genotyping. We studied 199 samples from patients originating from 26 countries and living in France. The HMA successfully genotyped 182 (91%) of these samples, as follows: 77 (42%) subtype A, 57 (31%) subtype B, 5 (3%) subtype C, 5 (3%) subtype D, 8 (4%) subtype E, 22 (12%) subtype F, 5 (3%) subtype G, and 3 (2%) subtype H. We were not able to genotype 12 samples by means of the HMA. These latter strains were sequenced, and phylogenetic analyses revealed that they were highly divergent subtype A-, D-, or G-related strains. Eight (of 12) subtype D strains were indeterminate by HMA, owing to the broad intrasubtype diversity, suggesting that new reference subtype D plasmids are required, as previously proposed. Thirty-seven strains belonging to the different subtypes were sequenced, and the results showed perfect concordance with the HMA results. Interlaboratory quality controls confirmed the reliability of the HMA for HIV-1 subtyping, despite the extensive viral variability. However, plasmid selection must be continuously revised to cover viral diversification.

Multiply spliced env and nef transcripts of simian immunodeficiency virus from West African green monkey (SIVagm-sab).

We have characterized the spliced transcripts of nef and envelope genes of SIVagm from African green monkey of the sabaeus subspecies. Most of the transcripts we have studied, representing the most abundant mRNA species in our assay, have undergone a specific splicing event that removes a part of the trans-activation response (TAR) element. This region is predicted to form a stable secondary structure (four stem-loop elements in SIVagm-sab) that affects the trans-activation of viral gene expression by Tat and the translation of the viral transcripts. Contrary to what is observed in other viruses, in which this R-region splicing has also been described (e.g., HIV-2), the LTR splicing in SIVagm-sab removes part of the first stem-loop and the following ones, nearly completely disrupting the TAR element secondary structure. Because LTR splicing seems to be a conserved feature among the strains we have characterized, these results suggest that this phenomenon could have important consequences for virus replication, pathogenicity, and latency.

Genetic subtypes of HIV type 1 and HIV type 2 strains in commercial sex workers from Bamako, Mali.

In Africa the highest HIV infection rate has been reported among female commercial sex workers (CSWs) who are at increasing risk of acquiring and transmitting HIV infection. In October 1995, 176 CSWs were studied in Bamako, the capital city of Mali. The ages of the CSWs ranged from 15 to 50 years old (mean, 28.8 years). Only 20.45% of the 176 CSWs were Malian; the majority were from Nigeria (32.9%) and Ghana (31.8%), and the remaining were from other African countries. Forty-one percent were active for less than 1 year as a commercial sex worker, and the length of prostitution for the remaining women ranged from 1 to 15 years (mean, 2.76). A total of 81 (46.02%) of the 176 CSWs were positive for HIV antibodies; 63 (35.8%) were HIV-1 positive, (3.9%) were HIV-2 positive, 11 (6.2%) had antibodies to HIV-1 and HIV-2, and none of them had antibodies to group O viruses. For all HIV antibody-positive samples, PBMCs were separated and genetic subtypes of HIV-1 were determined using the heteroduplex mobility assay (HMA), with ED5-ED12 as outer and ES7-ES8 as inner primers. Among the 66 HIV-1 strains characterized, 53 (80.3%) were subtype A, 2 (3.1%) belonged to subtype C, 1 (1.5%) belonged to subtype D, and 10 (15.1%) were identified as subtype G. Among the 10 subtype G strains, 8 were obtained from women who were very recent CSWs, with an activity of 1 year or less, assuming that there is a high probability that these infections occurred recently. Genetic subtypes of five HIV-2 viruses were determined by sequencing of the env and/or gag genes followed by phylogenetic analysis, and all of them belonged to subtype A. Comparison of HIV-1 and HIV-2 seroprevalence data from our study with previous data from Mali shows a significant rise in HIV-1 prevalence and a significant decrease in HIV-2 prevalence and confirms similar trends observed in neighboring countries. We have found four different genetic subtypes of HIV-1; however, subtype A is predominant and accounts for 80% of the cases and 15% of the HIV-1 infections were subtype G. It is important to continue the surveillance of subtypes on a systematic basis in order to see to what extent the proportions of the different subtypes will change over time.

PIP: The genetic variability of HIV-1 was investigated in a 1995 study of 176 commercial sex workers (CSWs) recruited in different areas in Bamako, Mali. 36 CSWs (20.45%) were born in Mali; 58 (32.9%) were from Nigeria and 56 (31.8%) were from Ghana. They ranged in age from 15-50 years (mean, 28.8 years). 41% of sex workers had been active for less than 12 months; the remaining women had been CSWs for 1-15 years (mean, 2.76 years). Of the 81 CSWs (46.02%) who were HIV-positive, 63 (35.8%) were infected with HIV-1, 7 (3.9%) with HIV-2, and the remaining 11 (6.2%) had antibodies to both HIV-1 and HIV-2. In contrast to other studies conducted among CSWs in Africa, none of these sex workers had antibodies to group O viruses. HIV-1 prevalence increased with age and length of time in prostitution and was higher among women with a history of sexually transmitted diseases. Among the 66 HIV-1 strains characterized, 53 (80.3%) were subtype A, 2 (3.1%) belonged to subtype C, 1 (1.5%) belonged to subtype D, and 10 (15.1%) were identified as subtype G. These results indicate a significant rise in HIV-1 prevalence and significant decreases in HIV-2 and combined HIV-1 and HIV-2 prevalence (10%, 15%, and 13%, respectively, in 1985). Ongoing surveillance of HIV-1 subtypes in Africa is important to identify shifts in the proportions of different subtypes over time. The genetic diversity of HIV has important implications for vaccine development.