Genome-Wide Divergence in the West-African Malaria Vector Anopheles melas.

Anopheles melas is a member of the recently diverged An. gambiae species complex, a model for speciation studies, and is a locally important malaria vector along the West-African coast where it breeds in brackish water. A recent population genetic study of An. melas revealed species-level genetic differentiation between three population clusters. An. melas West extends from The Gambia to the village of Tiko, Cameroon. The other mainland cluster, An. melas South, extends from the southern Cameroonian village of Ipono to Angola. Bioko Island, Equatorial Guinea An. melas populations are genetically isolated from mainland populations. To examine how genetic differentiation between these An. melas forms is distributed across their genomes, we conducted a genome-wide analysis of genetic differentiation and selection using whole genome sequencing data of pooled individuals (Pool-seq) from a representative population of each cluster. The An. melas forms exhibit high levels of genetic differentiation throughout their genomes, including the presence of numerous fixed differences between clusters. Although the level of divergence between the clusters is on a par with that of other species within the An. gambiae complex, patterns of genome-wide divergence and diversity do not provide evidence for the presence of pre- and/or postmating isolating mechanisms in the form of speciation islands. These results are consistent with an allopatric divergence process with little or no introgression.

Increasing outdoor host-seeking in Anopheles gambiae over 6 years of vector control on Bioko Island.

BACKGROUND: Vector control through indoor residual spraying (IRS) has been employed on Bioko Island, Equatorial Guinea, under the Bioko Island Malaria Control Project (BIMCP) since 2004. This study analyses the change in mosquito abundance, species composition and outdoor host-seeking proportions from 2009 to 2014, after 11 years of vector control on Bioko Island. METHODS: All-night indoor and outdoor human landing catches were performed monthly in the Bioko Island villages of Mongola, Arena Blanca, Biabia and Balboa from 2009 to 2014. Collected mosquitoes were morphologically identified and a subset of Anopheles gambiae sensu lato (s.l.) were later identified molecularly to their sibling species. Mosquito collection rates, species composition and indoor/outdoor host-seeking sites were analysed using generalized linear mixed models to assess changes in mosquito abundance and behaviour. RESULTS: The overall mosquito collection rate declined in each of the four Bioko Island villages. Anopheles coluzzii and Anopheles melas comprised the An. gambiae s.l. mosquito vector population, with a range of species proportions across the four villages. The proportion of outdoor host-seeking An. gambiae s.l. mosquitoes increased significantly in all four villages with an average increase of 58.8 % [57.9, 59.64 %] in 2009 to 70.0 % [67.8, 72.0 %] in 2014. Outdoor host-seeking rates did not increase in the month after an IRS spray round compared to the month before, suggesting that insecticide repellency has little impact on host-seeking behaviour. CONCLUSION: While vector control on Bioko Island has succeeded in substantial reduction in overall vector biting rates, populations of An. coluzzii and An. melas persist. Host-seeking behaviour has changed in these An. gambiae s.l. populations, with a shift towards outdoor host-seeking. During this study period, the proportion of host-seeking An. gambiae s.l. caught outdoors observed on Bioko Island increased to high levels, exceeding 80 % in some locations. It is possible that there may be a genetic basis underlying this large shift in host-seeking behaviour, in which case outdoor feeding could pose a serious threat to current vector control programmes. Currently, the BIMCP is preparing for this potential challenge by testing source reduction as a complementary control effort that also targets outdoor transmission.

Five years of malaria control in the continental region, Equatorial Guinea.

BACKGROUND: A successful malaria control programme began in 2004 on Bioko Island, Equatorial Guinea. From 2007, the same multiple malaria interventions, though reduced in scope for funding reasons, were introduced to the four mainland provinces of Equatorial Guinea (the continental region) aiming to recreate Bioko's success. Two provinces received long-lasting insecticidal nets (LLINs) and two provinces received biannual indoor residual spraying (IRS). Enhanced case management and communications were introduced throughout. METHODS: Estimates of intervention coverage and indicators of malaria transmission for 2007 to 2011 were derived from annual malaria indicator surveys (MIS). Results were complemented by health information system (HIS) and entomological data. The personal protection offered by LLINs and IRS against Plasmodium falciparum infection was estimated with logistic regression. RESULTS: The estimated proportion of children aged 1-4 using either an LLIN the previous night or living in a house sprayed in the last six months was 23% in 2007 and 42% in 2011. The estimated prevalence of P. falciparum in children aged 1-4 was 68% (N=1,770; 95% confidence interval [CI]: 58-76%) in 2007 and 52% (N=1,602; 95% CI: 44-61%) in 2011. Children 1-4 years had lower prevalence if they used an LLIN the previous night (N=1,124, 56%; adjusted odds ratio [aOR] 0.64, 95% CI: 0.55-0.74) or if they lived in a sprayed house (N=1,150, 57%; aOR 0.80, 95% CI: 0.62-1.03) compared to children with neither intervention (N=4,131, 66%, reference group). The minority of children who both used an LLIN and lived in a sprayed house had the lowest prevalence of infection (N=171, 45%; aOR 0.52, 95% CI: 0.35-0.78). High site-level intervention coverage did not always correlate with lower site-level P. falciparum prevalence. The malaria season peaked in either June or July, not necessarily coinciding with MIS data collection. CONCLUSIONS: Though moderate impact was achieved after five years of vector control, case management, and communications, prevalence remained high due to an inability to sufficiently scale-up coverage with either IRS or LLINs. Both LLINs and IRS provided individual protection, but greater protection was afforded to children who benefitted from both.

Country-level operational implementation of the Global Plan for Insecticide Resistance Management.

Malaria control is reliant on the use of long-lasting pyrethroid-impregnated nets and/or indoor residual spraying (IRS) of insecticide. The rapid selection and spread of operationally significant pyrethroid resistance in African malaria vectors threatens our ability to sustain malaria control. Establishing whether resistance is operationally significant is technically challenging. Routine monitoring by bioassay is inadequate, and there are limited data linking resistance selection with changes in disease transmission. The default is to switch insecticides when resistance is detected, but limited insecticide options and resistance to multiple insecticides in numerous locations make this approach unsustainable. Detailed analysis of the resistance situation in Anopheles gambiae on Bioko Island after pyrethroid resistance was detected in this species in 2004, and the IRS program switched to carbamate bendiocarb, has now been undertaken. The pyrethroid resistance selected is a target-site knock-down resistance kdr-form, on a background of generally elevated metabolic activity, compared with insecticide-susceptible A. gambiae, but the major cytochrome P450-based metabolic pyrethroid resistance mechanisms are not present. The available evidence from bioassays and infection data suggests that the pyrethroid resistance mechanisms in Bioko malaria vectors are not operationally significant, and on this basis, a different, long-lasting pyrethroid formulation is now being reintroduced for IRS in a rotational insecticide resistance management program. This will allow control efforts to be sustained in a cost-effective manner while reducing the selection pressure for resistance to nonpyrethroid insecticides. The methods used provide a template for evidence-based insecticide resistance management by malaria control programs.

Insecticide resistance allele frequencies in Anopheles gambiae before and after anti-vector interventions in continental Equatorial Guinea.

Anti-malaria interventions that rely on insecticides can be compromised by insecticide-resistance alleles among malaria vectors. We examined frequency changes of resistance alleles at two loci, knockdown resistance (kdr) and acetylcholinesterase-1 (ace-1), which confer resistance to pyrethroids and DDT, and carbamates, respectively. A total of 7,059 Anopheles gambiae sensu stricto mosquitoes were analyzed from multiple sites across continental Equatorial Guinea. A subset of sites included samples collected pre-intervention (2007) and post-intervention (2009-2011). Both L1014S and L1014F resistance alleles were observed in almost all pre-intervention collections. In particular, L1014F was already at substantial frequencies in M form populations (17.6-74.6%), and at high frequencies (> 50%) in all but two S form populations. Comparison before and throughout anti-vector interventions showed drastic increases in L1014F, presumably caused by intensified selection pressure imposed by pyrethroids used in vector control efforts. In light of these findings, inclusion of other insecticide classes in any anti-vector intervention can be considered prudent.

Large fluctuations in the effective population size of the malaria mosquito Anopheles gambiae s.s. during vector control cycle.

On Bioko Island, Equatorial Guinea, indoor residual spraying (IRS) has been part of the Bioko Island Malaria Control Project since early 2004. Despite success in reducing childhood infections, areas of high transmission remain on the island. We therefore examined fluctuations in the effective population size (N e ) of the malaria vector Anopheles gambiae in an area of persistent high transmission over two spray rounds. We analyzed data for 13 microsatellite loci from 791 An. gambiae specimens collected at six time points in 2009 and 2010 and reconstructed the demographic history of the population during this period using approximate Bayesian computation (ABC). Our analysis shows that IRS rounds have a large impact on N e , reducing it by 65%-92% from prespray round N e . More importantly, our analysis shows that after 3-5 months, the An. gambiae population rebounded by 2818% compared shortly following the spray round. Our study underscores the importance of adequate spray round frequency to provide continuous suppression of mosquito populations and that increased spray round frequency should substantially improve the efficacy of IRS campaigns. It also demonstrates the ability of ABC to reconstruct a detailed demographic history across only a few tens of generations in a large population.

The effective population size of malaria mosquitoes: large impact of vector control.

Malaria vectors in sub-Saharan Africa have proven themselves very difficult adversaries in the global struggle against malaria. Decades of anti-vector interventions have yielded mixed results--with successful reductions in transmission in some areas and limited impacts in others. These varying successes can be ascribed to a lack of universally effective vector control tools, as well as the development of insecticide resistance in mosquito populations. Understanding the impact of vector control on mosquito populations is crucial for planning new interventions and evaluating existing ones. However, estimates of population size changes in response to control efforts are often inaccurate because of limitations and biases in collection methods. Attempts to evaluate the impact of vector control on mosquito effective population size (N(e)) have produced inconclusive results thus far. Therefore, we obtained data for 13-15 microsatellite markers for more than 1,500 mosquitoes representing multiple time points for seven populations of three important vector species--Anopheles gambiae, An. melas, and An. moucheti--in Equatorial Guinea. These populations were exposed to indoor residual spraying or long-lasting insecticidal nets in recent years. For comparison, we also analyzed data from two populations that have no history of organized vector control. We used Approximate Bayesian Computation to reconstruct their demographic history, allowing us to evaluate the impact of these interventions on the effective population size. In six of the seven study populations, vector control had a dramatic impact on the effective population size, reducing N(e) between 55%-87%, the exception being a single An. melas population. In contrast, the two negative control populations did not experience a reduction in effective population size. This study is the first to conclusively link anti-vector intervention programs in Africa to sharply reduced effective population sizes of malaria vectors.

Malaria transmission after five years of vector control on Bioko Island, Equatorial Guinea.

BACKGROUND: Malaria is endemic with year-round transmission on Bioko Island. The Bioko Island Malaria Control Project (BIMCP) started in 2004 with the aim to reduce malaria transmission and to ultimately eliminate malaria. While the project has been successful in reducing overall malaria morbidity and mortality, foci of high malaria transmission still persist on the island. Results from the 2009 entomological collections are reported here. METHODS: Human landing collections (HLC) and light trap collections (LTC) were carried out on Bioko Island, Equatorial Guinea in 2009. The HLCs were performed in three locations every second month and LTCs were carried out in 10 locations every second week. Molecular analyses were performed to identify species, detect sporozoites, and identify potential insecticide resistance alleles. RESULTS: The entomological inoculation rates (EIR) on Bioko Island ranged from 163 to 840, with the outdoor EIRs reaching > 900 infective mosquito bites per year. All three human landing collection sites on Bioko Island had an annual EIR exceeding the calculated African average of 121 infective bites per year. The highest recorded EIRs were in Punta Europa in northwestern Bioko Island with human biting rates of 92 and 66 mosquito landings per person per night, outdoors and indoors, respectively. Overall, the propensity for mosquito biting on the island was significantly higher outdoors than indoors (p < 0.001). Both Anopheles gambiae s.s. and An. melas were responsible for malaria transmission on the island, but with different geographical distribution patterns. Sporozoite rates were the highest in An. gambiae s.s. populations ranging from 3.1% in Punta Europa and 5.7% in Riaba in the southeast. Only the L1014F (kdr-west) insecticide resistance mutation was detected on the island with frequencies ranging from 22-88% in An. gambiae s.s. No insecticide resistance alleles were detected in the An. melas populations. CONCLUSIONS: In spite of five years of extensive malaria control and a generalized reduction in the force of transmission, parasite prevalence and child mortality, foci of very high transmission persist on Bioko Island, particularly in the northwestern Punta Europa area. This area is favorable for anopheline mosquito breeding; human biting rates are high, and the EIRs are among the highest ever recorded. Both vector species collected in the study have a propensity to bite outdoors more frequently than indoors. Despite current vector control efforts mosquito densities remain high in such foci of high malaria transmission. To further reduce transmission, indoor residual spraying (IRS) needs to be supplemented with additional vector control interventions.

Multicentre studies of insecticide-treated durable wall lining in Africa and South-East Asia: entomological efficacy and household acceptability during one year of field use.

BACKGROUND: Indoor residual spraying (IRS) is a primary method of malaria vector control, but its potential impact is constrained by several inherent limitations: spraying must be repeated when insecticide residues decay, householders can tire of the annual imposition and campaign costs are recurrent. Durable lining (DL) can be considered an advanced form of long-lasting IRS where insecticide is gradually released from an aesthetically attractive wall lining material to provide vector control for several years. A multicentre trial was carried out in Equatorial Guinea, Ghana, Mali, South Africa and Vietnam to assess the feasibility, durability, bioefficacy and household acceptability of DL, compared to conventional IRS or insecticide-treated curtains (LLITCs), in a variety of operational settings. METHODS: This study was conducted in 220 households in traditional rural villages over 12-15 months. In all sites, rolls of DL were cut to fit house dimensions and fixed to interior wall surfaces (usually with nails and caps) by trained teams. Acceptability was assessed using a standardized questionnaire covering such topics as installation, exposure reactions, entomology, indoor environment, aesthetics and durability. Bioefficacy of interventions was evaluated using WHO cone bioassay tests at regular intervals throughout the year. RESULTS: The deltamethrin DL demonstrated little to no decline in bioefficacy over 12-15 months, supported by minimal loss of insecticide content. By contrast, IRS displayed a significant decrease in bioactivity by 6 months and full loss after 12 months. The majority of participants in DL households perceived reductions in mosquito density (93%) and biting (82%), but no changes in indoor temperature (83%). Among those households that wanted to retain the DL, 73% cited protective reasons, 20% expressed a desire to keep theirs for decoration and 7% valued both qualities equally. In Equatorial Guinea, when offered a choice of vector control product at the end of the trial (DL, IRS or LLITCs), DL consistently emerged as the most popular intervention regardless of the earlier household allocation. CONCLUSIONS: Just as long-lasting insecticidal nets overcame several of the technical and logistical constraints associated with conventionally treated nets and then went to scale, this study demonstrates the potential of DL to sustain user compliance and overcome the operational challenges associated with IRS.

Genetic isolation within the malaria mosquito Anopheles melas.

Anopheles melas is a brackish water-breeding member of the Anopheles gambiae complex that is distributed along the coast of West Africa and is a major malaria vector within its range. Because little is known about the population structure of this species, we analysed 15 microsatellite markers and 1161 bp of mtDNA in 11 A. melas populations collected throughout its range. Compared with its sibling species A. gambiae, A. melas populations have a high level of genetic differentiation between them, representing its patchy distribution due to its fragmented larval habitat that is associated with mangroves and salt marsh grass. Populations clustered into three distinct groups representing Western Africa, Southern Africa and Bioko Island populations that appear to be mostly isolated. Fixed differences in the mtDNA are present between all three clusters, and a Bayesian clustering analysis of the microsatellite data found no evidence for migration from mainland to Bioko Island populations, and little migration was evident between the Southern to the Western cluster. Surprisingly, mtDNA divergence between the three A. melas clusters is on par with levels of divergence between other species of the A. gambiae complex, and no support for monophyly was observed in a maximum-likelihood phylogenetic analysis. Finally, an approximate Bayesian analysis of microsatellite data indicates that Bioko Island A. melas populations were connected to the mainland populations in the past, but became isolated, presumably when sea levels rose after the last glaciation period (≥10 000-11 000 bp). This study has exposed species-level genetic divergence within A. melas and also has implications for control of this malaria vector.

Increased risks of malaria due to limited residual life of insecticide and outdoor biting versus protection by combined use of nets and indoor residual spraying on Bioko Island, Equatorial Guinea.

BACKGROUND: Malaria is endemic on Bioko Island, Equatorial Guinea, with year-round transmission. In 2004 an intensive malaria control strategy primarily based on indoor residual spraying (IRS) was launched. The limited residual life of IRS poses particular challenges in a setting with year-round transmission, such as Bioko. Recent reports of outdoor biting by Anopheles gambiae are an additional cause for concern. In this study, the effect of the short residual life of bendiocarb insecticide and of children spending time outdoors at night, on malaria infection prevalence was examined. METHODS: Data from the 2011 annual malaria indicator survey and from standard WHO cone bioassays were used to examine the relationship between time since IRS, mosquito mortality and prevalence of infection in children. How often children spend time outside at night and the association of this behaviour with malaria infection were also examined. RESULTS: Prevalence of malaria infection in two to 14 year-olds in 2011 was 18.4%, 21.0% and 28.1% in communities with median time since IRS of three, four and five months respectively. After adjusting for confounders, each extra month since IRS corresponded to an odds ratio (OR) of 1.44 (95% CI 1.15-1.81) for infection prevalence in two to 14 year-olds. Mosquito mortality was 100%, 96%, 81% and 78%, at month 2, 3, 4 and 5 respectively after spraying. Only 4.1% of children spent time outside the night before the survey between the hours of 22.00 and 06.00 and those who did were not at a higher risk of infection (OR 0.87, 95% CI 0.50-1.54). Sleeping under a mosquito net provided additive protection (OR 0.68, 95% CI 0.54-0.86). CONCLUSIONS: The results demonstrate the epidemiological impact of reduced mosquito mortality with time since IRS. The study underscores that in settings of year-round transmission there is a compelling need for longer-lasting IRS insecticides, but that in the interim, high coverage of long-lasting insecticidal nets (LLINs) may ameliorate the loss of effect of current shorter-lasting IRS insecticides. Moreover, continued use of IRS and LLINs for indoor-oriented vector control is warranted given that there is no evidence that spending time outdoors at night increases infection prevalence in children.

Light traps fail to estimate reliable malaria mosquito biting rates on Bioko Island, Equatorial Guinea.

BACKGROUND: The human biting rate (HBR), an important parameter for assessing malaria transmission and evaluating vector control interventions, is commonly estimated by human landing collections (HLC). Although intense efforts have been made to find alternative non-exposure mosquito collection methods, HLC remains the standard for providing reliable and consistent HBRs. The aim of this study was to assess the relationship between human landing and light trap collections (LTC), in an attempt to estimate operationally feasible conversion factors between the two. The study was conducted as part of the operational research component of the Bioko Island Malaria Control Project (BIMCP), Equatorial Guinea. METHODS: Malaria mosquitoes were collected indoors and outdoors by HLCs and LTCs in three villages on Bioko Island, Equatorial Guinea during five bimonthly collections in 2009. Indoor light traps were suspended adjacent to occupied long-lasting, insecticide-treated bed nets. Outdoor light traps were placed close to the outer wall under the roof of the collection house. Collected specimens were subjected to DNA extraction and diagnostic PCR to identify species within the Anopheles gambiae complex. Data were analysed by simple regression of log-transformed values and by Bayesian regression analysis. RESULTS: There was a poor correlation between the two collection methods. Results varied by location, venue, month, house, but also by the statistical method used. The more robust Bayesian analyses indicated non-linear relationships and relative sampling efficiencies being density dependent for the indoor collections, implying that straight-forward and simple conversion factors could not be calculated for any of the locations. Outdoor LTC:HLC relationships were weak, but could be estimated at 0.10 and 0.07 for each of two locations. CONCLUSIONS: Light trap collections in combination with bed nets are not recommended as a reliable method to assess human biting rates on Bioko Island. Different statistical analyses methods give variable and inconsistent results. Substantial variation in collection methods prevents the determination of reliable and operationally feasible conversion factors for both indoor and outdoor data. Until improved mosquito collection methods are developed that can provide reliable and unbiased HBR estimates, HLCs should continue to serve as the reference method for HBR estimation.

Determinants of bed net use in children under five and household bed net ownership on Bioko Island, Equatorial Guinea.

BACKGROUND: As part of comprehensive malaria control strategies, the Bioko Island Malaria Control Project (BIMCP) distributed 110,000 long-lasting insecticide-treated nets (LLIN) in late 2007 with the aim of providing one net for each sleeping area. Despite attaining initially very high levels of net coverage and net use, many children under five years of age did not sleep under a net by 2009, according to annual malaria indicator surveys. The aim of this study was to assess the determinants of bed net use in children under five and bed net ownership of the households in which they live. METHODS: Using data from annual cross-sectional household surveys of 2008 and 2009, we investigated factors associated with sleeping under a mosquito net the night prior to the survey, and a households owning at least one net, in all households which had at least one child under five years. Amongst others, caregiver's knowledge of malaria and household characteristics including a socio-economic score (SES), based on ownership of household assets, were analysed for their effect on net ownership and use. RESULTS: There was a decline of around 32% in the proportion of households that owned at least one net between 2008 and 2009. Higher household bed net ownership was associated with knowing how malaria was prevented and transmitted, having the house sprayed in the previous 12 months, having fewer children under five in the household, and children being sick at some point in the previous 14 days. Higher bed net use in children < 5 was associated with being sick at some point in the last 14 days prior to the survey, living in an urban area, more years of education of the head of the household, household ownership of at least one ITN (as opposed to an untreated net) and the year in which the survey took place. CONCLUSIONS: The big fall in bed net use from 2008 to 2009 was attributable to the striking decline in ownership. Although ownership was similar in rural and urban areas, rural households were less likely to protect their children with bed nets. Knowledge about malaria was an important determinant of bed net ownership. Further research is needed to elucidate the decline in bed net ownership between 2008 and 2009.

How much does malaria vector control quality matter: the epidemiological impact of holed nets and inadequate indoor residual spraying.

BACKGROUND: Insecticide treated nets (ITN) and indoor residual spraying (IRS) are the two pillars of malaria vector control in Africa, but both interventions are beset by quality and coverage concerns. Data from three control programs were used to investigate the impact of: 1) the physical deterioration of ITNs, and 2) inadequate IRS spray coverage, on their respective protective effectiveness. METHODS: Malaria indicator surveys were carried out in 2009 and 2010 in Bioko Island, mainland Equatorial Guinea and Malawi to monitor infection with P. falciparum in children, mosquito net use, net condition and spray status of houses. Nets were classified by their condition. The association between infection and quality and coverage of interventions was investigated. RESULTS: There was reduced odds of infection with P. falciparum in children sleeping under ITNs that were intact (Odds ratio (OR): 0.65, 95% CI: 0.55-0.77 and OR: 0.81, 95% CI: 0.56-1.18 in Equatorial Guinea and in Malawi respectively), but the protective effect became less with increasingly worse condition of the net. There was evidence for a linear trend in infection per category increase in deterioration of nets. In Equatorial Guinea IRS offered protection to those in sprayed and unsprayed houses alike when neighbourhood spray coverage was high (≥80%) compared to those living in areas of low IRS coverage (<20%), regardless of whether the house they lived in was sprayed or not (adjusted OR = 0.54, 95% CI 0.33-0.89). ITNs provided only personal protection, offering no protection to non users. Although similar effects were seen in Malawi, the evidence was much weaker than in Equatorial Guinea. CONCLUSIONS: Universal coverage strategies should consider policies for repair and replacement of holed nets and promote the care of nets by their owners. IRS programs should ensure high spray coverage since inadequate coverage gives little or no protection at all.

Morphometrical evidence for speciation within Bolivian Lutzomyia aragaoi (Diptera: Psychodidae).

For Bolivian specimens of Lutzomyia aragaoi (Costa Lima, A., 1932. Sobre os phlebotomos americanos (Diptera, Psychodidae). Mem. Inst. Oswaldo Cruz 26, 15-69), collected in the same light-traps, we show the existence of two distinct morphotypes distinguished by male parameres. These sympatric forms were observed in the Alto Beni, Subandean region of Bolivia, and in the North Beni, Amazonian region of Bolivia, two areas separated by large, sparsely forested, "llanos". Univariate morphometric comparisons between these two forms showed non-overlapping variation of size for external structures of the genitalia (lateral lobe, style and coxite), allowing separation of them on the basis of a single measurement. Multivariate discriminant analysis of head and wing dimensions-excluding genitalia structures-grouped the insects according to their morphotype instead of their geographic origins. The two forms also showed different patterns in number and distribution of low mesepisternal setae. The existence of these two separate forms in close sympatry suggests reproductive isolation, hence a speciation process.

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El INTERNET ahora es la mayor fuente de informacón en salud. Se registraron divestos sitios médicos en internet en español, ingles, frances y se presentan listas de direcciones URLs que se incolucran todas las areas de la salud, particularmente: Microbiologia, Parasitología y Ciencias Médicas. Estos sitios médicos en INTERNET proporcionan información alternativa en salud humana, particularmente para docentes, estudiantes en los niveles de pre y potgrado