Español (pdf)
Articulo en XML
Referencias del artículo
Traducción automática
Enviar articulo por email
Citado por SciELO 
Similares en
SciELO 
uBio 
Permalink
Boletín de Malariología y Salud Ambiental
versión impresa ISSN 1690-4648
Bol Mal Salud Amb vol.57 no.1 Maracay jul. 2017
Tasas de infección de Plasmodium spp. para algunos Anopheles spp. del municipio Sifontes, Estado Bolívar, Venezuela
Sandra Abou Orm 1-2 , Jorge E. Moreno 3 , Marifel Carrozza 1 , Porfirio Acevedo 3 & Flor Herrera 1*
1 Instituto de Investigaciones Biomédicas “Dr. Francisco Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo, Sede
Aragua, Maracay, Venezuela.
2 Departamento de Microbiología, Facultad de Ciencias de la Salud, Universidad de Carabobo, Sede Aragua, Maracay, Venezuela.
3 Centro de Investigaciones de Campo “Dr. Francesco Vitanza”, Servicio Autónomo Instituto de Altos Estudios “Dr. Arnoldo Gabaldon”, MPPS. Tumeremo, estado Bolívar, Venezuela.
*Autor de correspondencia: flormhq@gmail.com.
SUMMARY
Bolivar state represents the most important focus of malaria transmission in Venezuela, most malaria cases are related to gold-mining, especially in the Sifontes municipality. Therefore, the aim of this study was to determine the rate of natural Plasmodium spp. infection in Anopheles spp. collected between September 2009 and January 2012 in Sifontes. Out of 1,633 Anopheles spp. collected, the most abundant species were An. darlingi Root (56.7%), An. Albitarsis Lynch Arribalzaga sensulato(30.6%) and An. nuneztovari Gabaldon s.l. (11.7%). All the mosquitoes were assayed individually by the polymerase chain reaction (PCR) and sixty-five were positive for Plasmodium spp. An. albitarsis s.l. had the highest infection rate, 5.4% (27/499; 95% CI: 3.63- 7.76), followed by An. darlingi with 4.0% (37/926; 95% CI: 2.86-5.45) and An. nuneztovari s.l. with 0.5% (1/191; 95% CI: 0.03-2.58). The overall infection rate was 3.9% (65/1,633; 95% CI:3.10-5.0). The rates of infection by Plasmodium species in mosquitoes were 3.7% (61/1633) for P. vivax and 0.2% (4/1633) for P. falciparum. The results show that the species An. albitarsis s.l.increased its rate of infection by Plasmodium more than 3 times in ten years. This natural infection of the vectors by Plasmodium may explain the current epidemiological situation of the Sifontes municipality as not only would the transmission remain, it would also be promoted.
Key words: Malaria, natural infection, Plasmodium spp., Anopheles darlingi, An. albitarsis s.l., Sifontes Municipality.
Plasmodium spp. infection rates for some Anopheles spp. from Sifontes Municipality, Bolívar State, Venezuela
RESUMEN
El Estado Bolívar representa el foco más importante de transmisión de malaria en Venezuela; la mayoría de los casos están relacionados a la minería de oro, especialmente en el municipio Sifontes. Por lo tanto, el objetivo de este estudio fue determinar la tasa de infección natural de Plasmodium spp. en Anopheles spp. colectados entre septiembre de 2009 a enero de 2012 en Sifontes. De 1.633 Anopheles spp. colectados, las especies más abundantes fueron An. darlingi Root (56,7%), An. albitarsis Lynch Arribalzaga sensu lato(30,6%) y An. nunez tovari Gabaldon
s.l. (11,7%). La reacción en cadena de la polimerasa (PCR) se realizó individualmente para todos los mosquitos y sesenta y cinco resultaron positivos para Plasmodium spp.An. albitarsis s.l. tuvo la tasa de infección mayor, 5,4% (27/499; 95% CI: 3,63- 7,76), seguido por An. darlingi con 4,0% (37/926; 95% CI: 2,86-5,45) yAn.nunez tovari s.l. con 0,5% (1/191; 95% CI: 0,03-2,58).Las tasas de infección para las especies de Plasmodium en mosquitos fueron 3,7% (61/1633) para P. vivax y 0,2% (4/1633) para P.falciparum. Los resultados muestran que la especie An. albitarsis s.l. aumentó su tasa de infección por Plasmodium más de 3 veces en diez años. Esta infección natural de los vectores por Plasmodium podría explicar la situación epidemiológica actual del Municipio Sifontes la cual no sólo permitiría que la transmisión permaneciera sino que se promoviera.
Palabras clave: Malaria, infección natural, Plasmodium spp., Anopheles darlingi, An. albitarsis s.l., Municipio Sifontes.
Recibido el 29/04/2016 Aceptado el 22/03/2017
INTRODUCTION
Malaria is the most important parasitic disease in the world, the World Health Organization (WHO) has estimated that 214 million cases occurred in 2015 and 438,000 people died of malaria while more than 3.2 billion people are at risk of acquiring the disease worldwide (WHO, 2016). Latin America has made great efforts in the fight against malaria, though Venezuela is one of the few countries that fails to reduce the number of malaria cases, which could eventually endanger neighboring countries (WHO, 2016). Bolivar state represents the most important focus of malaria transmission in Venezuela and most malaria cases come from areas related to gold-mining, especially in the Sifontes municipality (Moreno et al., 2014). Evidence indicates that Anopheles darling Root and An. albitarsis s.l. are the principal vectors in the zone (Moreno et al., 2007, 2009; Rubio-Palis 2009). Researchers worldwide have claimed that rise in malaria is linking to deforestation and associated environmental changes (Pattanayak et al., 2006; Fornace et al., 2016). Deforestation can produce new habitats for some anopheline species as it can favor specific anopheline larvae development (Moreno et al., 2000, 2015a; Vittor et al., 2009; Medina et al., 2011).The increase in the number of Anopheles species increments the risk of human-mosquito contact and the risk of malaria infection. This is one of the main reasons why, in the Sifontes municipality where the deforestation is rising, the malaria transmission is quite significant.
The infection rates of Plasmodium species in Anopheles mosquito populations are necessary to provide estimates of the intensity of malaria transmission in a study area (Mahapatra et al., 2006; Rubio-Palis, 2009). The method considered the “gold standard” is the observation of sporozoites in the salivary glands of individual mosquitoes, but it is very laborious, time-consuming, requires fresh samples and does not distinguish among all the Plasmodium species (WHO, 1975). Two other methods have been used for estimating the sporozoite rate: The enzyme-linked immunoabsorbent assay (ELISA) using monoclonal antibodies targeting the circumsporozoite protein (Beier, 2002) and the Plasmodium-specific polymerase chain reaction (PCR) (Snounou, 1993; Rubio et al., 1999; Padley et al., 2003). In general, the PCR technique is more sensitive than ELISA, particularly with low levels of the salivary gland sporozoites.
In the Sifontes municipality, exists a high risk of contracting malaria. During 2016 177,619 out of 240,613 (73.8%) malaria cases were reported in Bolivar state, of which 102,543 (57.7%) comes from this municipality (DGSA, 2016). Therefore, the aim of this study was to determine the rate of natural Plasmodium spp. infection in Anopheles spp. collected between September 2009 and January 2012 in San Isidro parish of Sifontes municipality, activities conducted within the framework for malaria entomological surveillance and control of the Environmental Health Direction of the Bolivar State (DSAEB, 2012).
MATERIALS AND METHODS
Study Area
The study was performed in two areas with several localities of the San Isidro parish in Sifontes municipality (lat 6º00' - 7º54' N, long 60º44' - 61º39' W), Bolivar state, Venezuela. Moreno et al. (2014) already described the area.The municipality, according to the morbidity of malaria and their geographical distribution, has been divided into three foci: Tumeremo to the North, El Dorado in the middle and San Isidro to the South. In San Isidro parish, the first area “Las Claritas”, is formed by residential urban conglomerates of the communities of Las Claritas, Ciudad Dorada and several small towns located along a twenty kilometer transecta side road 10 (San Miguel de Betania, Las Manacas, Tierra Blanca, El Granzón, San Marco). The second area, adjacent to the former, is one of the largest deposits of gold of the country (Las Cristinas mine). Informal, small-scale miners actively mine these deposits resulting in significant deforestation through forest clearing. Both areas, despite the closeness, constitute two separate epidemiological stratus with different demographic and landscape features, and represent the focus with the highest malaria incidence over the last decade in Sifontes municipality as well as in Bolivar state (Moreno et al., 2014).
Mosquito collections
The female mosquitoes (1,633) were collected, both inside and outside houses, between September 2009-January 2012, monthly, 4-7 days in each month, using CDC light traps and up-draft ultra-violet traps (John W. Hock Co, Gainesville, FL), according to the protocol of Moreno et al. (2007). In the period August-December 2011 there was no mosquito collection, while in January 2012 it was only carried out in Las Cristinas. All Anopheles specimens were identified based on morphological features (Cova-García & Sutil, 1977; Faran & Linthicum, 1981; Rubio-Palis, 2000). After identification, groups of 5-10 Anopheles mosquitoes were stored in vials based on species, location and collection day, preserved in silica gel, transported to the Instituto de Investigaciones Biomédicas "Dr. Francisco Javier Triana Alonso” and stored at -80°C until their processing.
DNA extraction
The DNA was extracted from individual specimens using a phenol/chloroform method, resuspended in 50 μl of sterilized water and stored at
-80ºC (Rivero et al., 2004).
Nested PCR amplification
Detection and typing of Plasmodium spp. in Anopheles spp. was performed using a assay described by Snounou et al., (1993). This nested PCR assay uses Plasmodium genus-specific primers rPLU6 (5`-TTAAAATTGTTGCAGTTAAAACG-3`) and rPLU5 (5`-CCTGTTGTTGCCTTAAACTTC-3`) for the initial PCR amplification followed by species-specific primers for the second amplification: P. falciparum rFAL1 (5`-TTAAACTGGTTTGGGAAAACCAAATATATT-3`) and rFAL2 (5`-ACACAATGAACTCAATCATGACT ACCCGTC-3´), P. malariae rMAL1 (5´- ATA ACATAGTTGTACGTTAAGAATAACCGC-3´) and rMAL2 (5´- AAATTCCCATGCATAAAAAATTATACAAA-3´), P. vivax:
rVIV1 (5`- CGCTTCTAGCTTAATCCACATAACTGATAC-3´) and rVIV2 (5´-ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA-3´), P. ovaler OVA1 (5´-ATCTCTTTTGCTATTTTTTAGTATTGGAGA-3´) and rOVA2 (5´-GGAAAAGGACACATTAATTGTATCCTAGTG-3´). The PCR amplifications were carried out in 25 μl reaction volumes using 50 ng of template DNA in a PTC-100 thermal cycler (MJ Research, Inc., Watertown, MA). Negative controls (all reagents except template) were run to detect possible contamination. The amplified products were visualized by electrophoresis in 2% agarose gels stained with ethidium bromide.
Infection rate
The infection rate was estimated as the percentage of positive Plasmodium mosquitoes divided by the total number of mosquitoes assayed.
Data analysis
To estimate the infection rate, the mosquitoes collected were identified by species over all the villages and collection periods. To compare the observed differences in infection rates between anopheline species, the confidence intervals were calculated according to (Berry & Armitage, 1995). The program OpenEpi (Dean et al., 2015) was used for data analysis.
RESULTS
Anopheles spp. abundance
The distribution of mosquito species collected by period and locality is shown in Table 1. Of a total of 1,633 Anopheles spp. collected, the most abundant species were An. darlingi (56.70%), An. albitarsis s.l.(30.56%) and An. nuneztovari Gabaldon s.l. (11.70%). In 2010, more mosquitoes of all species were collected because the collection was all year round instead of certain months of the other years.
With respect to locality, An. albitarsis s.l. population was eight times (58.57%/7.19%) more abundant in Las Cristinas than in San Isidro while
An. darlingi and An. nuneztovari s.l. were 1.84 (72.23%/39.20%) and 12.79 (20.34%/1.59%) more abundant in San Isidro than in Las Cristinas during the period 2009-2011 (Table I). In January 2012, it was observed that the most abundant species in Las Cristinas was An. darlingi (47.25%) and An. albitarsis s.l. (39.01%). It was also noted that An. nuneztovari s.l. increased its population in that locality on the other years, almost 5 times (7.69%/1.59%). Moreover, An. triannulatus Neiva & Pinto s.l. went from having a very low abundance in both locations during the period 2009-2011 to 6% of abundance in 2012, representing an increase of 19 times in Las Cristinas (6.04%/0.32%).
Table I. Anopheles species composition, locality and period distribution in the Sifontes municipality.
Anopheles spp. Infection by Plasmodium spp.
The total of 1,633 mosquitoes were assayed individually by PCR (Table II). Sixty five werepositive for Plasmodium spp. Anopheles darlingi and
An. albitarsis s.l. were positive for P. vivax and P. falciparum, while An. nuneztovari s.l. was found to be only positive for P. vivax. Anopheles albitarsis s.l. had the highest infection rate, 5.41% (27/499; 95% CI: 3.63-7.76), followed by An. darlingi with 4.0% (37/926; 95% CI: 2.86-5.45) and An. nuneztovari s.l. with 0.52% (1/191; 95% CI:0.03-2.58).
The overall infection rate was 3.98% (65/1,633; 95% CI: 3.10-5.04) (Table 2). The rates of infection by Plasmodium species in mosquitoes were 3.7% (61/1633) for P. vivax and 0.2% (4/1633) for P. falciparum (Table II).
During the period September 2009 - July 2011, the highest rate of infection by Plasmodium spp. was in Las Cristinas with 6.4% (40/626; 95% CI: 4.56-8.52), distributed in An. darlingi 8.09% (20/247; 95% CI: 5.08-12.28), An. albitarsis s.l. 5.15% (19/369; 95% CI:3.19-7.89) and An. nuneztovari s.l. 10% (1/10; 95% CI: 0.5-49.32). In addition, a total of 3 An. triannulatus and 3 An. strodei were analyzed but none was infected. In San Isidro the rate of infection by Plasmodium spp. was 2.19% (18/821; 95% CI: 1.34-3.40) distributed in An. darlingi 2.36% (14/593; 95% CI: 1.34-3.87) and An. albitarsis s.l. 6.78% (4/59; 95% CI: 2.15-16.35). Here a total of 167 An. nuneztovari s.l., 1 An. triannulatus and 1 An. strodeiwere analyzed but none was infected (Table II). In January 2012, the collections were only in Las Cristinas and An. albitarsis s.l. had the highest infection rate by Plasmodium spp. with 5.63 % (4/71; 95% CI: 1.79-13.59) followed by An. darlingi with 3.5% (3/86; 95% CI: 0.88-9.49). Likewise, 14 An. nuneztovari s.l. and 11 An. triannulatus were analyzed but none was infected (Table II).
DISCUSSION
Anopheles albitarsis s.l. had a higher infection rate by Plasmodium spp. (5.4%) than An. darlingi (4.0%) for all months of the years studied, except in 2011 when it was collected no infected specimen, probably because An. albitarsis s.l. represented only 11.56% of the total mosquitoes collected, compared with 72.11% of An. darlingi. This result differs from Moreno et al., (2009) who worked in the same Sifontes municipality during 1999-2000 and reported a sporozoite rate ~ 3 times less for An. albitarsis s.l. (0.27%) than for An. darlingi (0.82%). It could mean that the species An. albitarsis s.l. had to increase its rate of infection by Plasmodium more than 3 times in ten years.
Table II. Natural Plasmodium infection in Anopheles spp. collected in Las Cristinas and San Isidro during Sept. 2009-Jan. 2012.
In spite, however, of the advantages of molecular diagnostic tests (most accuracy and sensitivity) to detect and identify the malaria parasite species, employment of these techniques for recognizing precisely infective stage mosquitoes and assessing sporozoite rates, needs to get rid of the abdomen of the mosquito. That is because if mosquitoes recently fed on human blood infected with malaria (erythrocytic form), false-positive results could be obtained. Here, in the present study, all positive Anopheles mosquitoes were considered to have infective sporozoites, and therefore the results could overestimate the true infection rate. However, it is important to be aware that this study gave emphasis to the relative difference between the infection rate of An. albitarsis s.l. and An. darlingi which could compensate for the overestimation of the absolute values and give a direct insight into the true scale of difference between both Anopheles.
It was shown that P. vivax infected more than P. falciparum the main Anopheles species present, 11.5 (3.67% P. vivax/0.32% P. falciparum) and 26.0 (5.21% P. vivax/0.20% P. falciparum) times more for An. darlingi and An. albitarsis s.l. respectively. This result was also greater than the one found in the Sifontes municipality a decade ago when the rate of infection of P. vivax was only 3.7 times more than P. falciparum (Moreno et al., 2009). Although both epidemiological and sampling situations are not comparable, this high natural infection of the vectors by Plasmodium may explain the current epidemiological situation of the Sifontes municipality.
Anopheles albitarsis s.l. was considered an opportunistic vector (Moreno et al., 2000, 2004, 2007) and had also been proposed as an emerging vector (Moreno et al., 2009) in Bolivar state. Although this study confirms that An. albitarsis s.l, and An. darlingi are primarily responsible for the malaria endemicity in the Sifontes municipality (Moreno et al., 2009), it demonstrates for the first time that An. albitarsis s.l. has a higher rate of Plasmodium infection than An. darlingi. These results suggest that An. albitarsis s.l. is gradually becoming the main species in Sifontes by displacing An. darlingi. However, further studies on the bionomics of these vectors are required to confirm this hypothesis.
Anopheles albitarsis s.l. has already been incriminated as a primary malaria vector in Amapá state of Brazil in a region of northeastern Amazonia (Conn et al., 2002). It was also reported that this species was more abundant than An. darlingi in Roraima, Brazil during the period 2001-2002 (Póvoa et al., 2006). The emergence of An. albitarsis s.l. was also informed in Puerto Carreño, Colombia and the report concluded that An. albitarsis s.l. with An. darlingi contribute to maintaining endemic malaria all year round in this region (Jiménez et al., 2012). At this point, it is important to note that, as with other species of the subgenus Nyssorhynchus, the taxon An. albitarsis s.l. is a complex of at least nine species morphologically indistinguishable from each other, of which only six have been formally described (Ruíz-López et al., 2012). At present, the bionomics and ecology of the species within this complex are not known with certainty (Ruíz-López et al., 2012). Of these, only An. albitarsis F has been confirmed in Bolivar state (Rubio-Palis et al., 2013).
The fact that An. albitarsis s.l. is a primary malaria vector in the Sifontes municipality may be due to deforestation from mining that impact the rainforest environment causing major ecological changes and favoring habitat for An. albitarsis s.l. Larvae of this species are common and abundant in man-made shallow water bodies exposed to sunlight, characteristic habitats generated by mining activities (Moreno et al., 2015a, 2015b). By contrast, An. darlingi is usually the predominant species in environments partially shaded, near the borders of forests with high humidity and rainfall (Bertiet al., 2008, Moreno et al., 2015a, 2015b). It has been reported that in Ramal do Granada, Brazil, populations of An. darlingi decreased depending on deforestation that is caused by human activity (Moutinho et al., 2011). On the other hand, the spatial distribution of An. albitarsis s.l. in the Atlantic forest in southeastern Brazil, correlated with open areas or deforested lands which are associated with changes in the natural ecosystem (Laporta et al., 2011). Also, in this case, a significant association between the presence of An. albitarsis s.l. with malaria cases was found.
During the study period (September 2009-January 2012), the Sifontes municipality reported 60,073 accumulative malaria cases which represented 54.5% of the nationwide malaria cases and 42,748 (71.1%) were registered in San Isidro parish (DSAEB, 2009-2012). In this parish, the area of Las Claritas, very deforested for being a mining town (Las Cristinas mine) located at km 88, was the one with the highest prevalence of An. albitarsis s.l. (54%) compared to An. darlingi (41%). These ecological changes that favor An. albitarsis s.l. habitats together with the massive convergence of people from different parts of Venezuela and from other countries who can carry the parasite, suggest that malaria transmission will continue to increase, especially if the natural vegetation continues reducing in the municipality. To clarify this point, it would be necessary to study the dynamics of malaria incidence and of the vector populations between partially and totally deforested areas to determine the relationships among variables under the two conditions.
Other species present in the Sifontes municipality were An. nuneztovari s.l., predominant vector in western Venezuela (Rubio-Palis, 2009), and also, albeit to a lesser extent, An. triannulatus s.l. which is considered a secondary vector (Sinka et al., 2010), although recently there was reported that it was one of the most abundant in Colombia (Rosero et al., 2013).
A key element of the malaria prevention and control is the surveillance of mosquito populations. This includes various factors like the mosquito
population abundance, Anopheles and Plasmodium species circulating in a certain area, and the Anopheles infection rates, especially in areas in which there are several Anopheles species that can transmit malaria, like the Sifontes municipality (Galardo et al., 2007).The precise determination of this last factor, according to the results of this study, should be considered to strengthen public health laboratories and reorient strategies for controlling malaria vectors in the Sifontes municipality.
ACKNOWLEDGEMENTS
To Dr. Carlos Espino for helping us in the statistic calculation; to the staff of the Centro de Investigaciones de Campo “Dr. Francesco Vitanza”, Tumeremo, estado Bolívar, Venezuela. Servicio Autónomo Instituto de Altos Estudios “Dr. Arnoldo Gabaldon”, MPPS for mosquito collections.
Conflict of interest
The authors declare that they have no conflict of interest.
REFERENCES
1.Beier J. (2002). Vector Incrimination and Entomological Inoculation Rates. pp. 3-11. In: Methods in Molecular Medicine, Vol. 72: Malaria Methods and Protocols. Edited by: Doolan D. © Humana Press, Inc., Totowa, NJ, [ Links ] USA.
2.Berry G. & Armitage P. (1995). Mid-P confidence intervals: a brief review. Statistician. 44: 417-423. [ Links ]
3.Berti J., Gonzáles J. & Navarro E. (2008). Fluctuaciones estacionales y temporales de la densidad larvaria de Anopheles darlingi Root (Diptera: Culicidae) y familias de insectos asociados al hábitat en El Granzón, Parroquia San Isidro, municipio Sifontes del estado Bolívar, Venezuela. Bol. Mal. Salud Amb. 48: 177-189. [ Links ]
4.Conn J., Wilkerson R., Segura M., De Souza R. & Schlichting C., Wirtz R. et al.(2002). Emergence of a new neotropical malaria vector facilitated by human migration and changes in land use. Am. J. Trop. Med. Hyg. 66: 18-22. [ Links ]
5.Cova-García P. & Sutil E. (1977). Claves gráficas para la clasificación de anofelinos de Venezuela. Publicaciones de la División de Endemias Rurales. Dirección de Malariología y Saneamiento Ambiental, MSAS. Maracay, Venezuela. [ Links ]
6.Dean A .G., Sullivan K. M. & Soe M. M. (2015). OpenEpi: Open Source Epidemiologic Statistics for Public Health [Software]. Available from:
www.OpenEpi.com.
7.Dirección General de Salud Ambiental. Dirección Vigilancia Epidemiológica MPPS (2016). Maracay, Venezuela. [ Links ]
8.Dirección General de Salud Ambiental (2012). Boletín Integral de Salud Ambiental. (Semana epidemiológica Nº 52, Año 18). Maracay, Venezuela. [ Links ]
9.Faran M. & Linthicum K. (1981). A handbook of the Amazonian species of Anopheles (Nyssorhynchus) (Díptera: Culicidae). Mosq. Syst. 13: 1-81. [ Links ]
10.Fornace K. M., Abidin T. R., Alexander N., Brock P., Grigg M. J., Murphy A., et al. (2016). Association between landscape factors and spatial patterns of Plasmodium knowlesi infections in Sabah, Malaysia. Emerg. Infect. Dis. 22: 201- 209. [ Links ]
11.Galardo A., Arruda M., Couto A., Wirtz R., Lounibos L. & Zimmerman R. (2007). Malaria vector incrimination in three rural riverine villages in the Brazilian Amazon. Am. J. Trop. Med. Hyg. 76: 461-469. [ Links ]
12.Jiménez P., Conn J., Wirtzand R. & Brochero H. (2012). Anopheles (Diptera: Culicidae) malaria vectors in themunicipality of Puerto Carreño,
Vichada, Colombia. Biomedica. 32: 13-21.
13.Laporta G. Z., Ramos D. G., Ribeiro M. C. & Sallum M. A. (2011). Habitat suitability of Anopheles vector species and association with human malaria in the Atlantic Forest in south-eastern Brazil. Mem.Inst. Oswaldo Cruz. 106 (Suppl. I): 239-245. [ Links ]
14.Mahapatra N., Marai N., Ranjit M., Parida S., Hansdah D., Hazra R., et al. (2006). Detection of Plasmodium falciparum infection in Anopheles mosquitos from Keonjhar district, Orissa, India. J. Vect. Borne Dis. 43: 191-194. [ Links ]
15.Medina D., Bevilacqua M., Cárdenas L., Morales L., Rubio-Palis Y., Martínez A., et al. (2011). Mapa de riesgo de transmisión de malaria en la cuenca del río Caura, Venezuela. Bol. Mal. Sal. Amb. 51:129-144. [ Links ]
16.Moreno J., Rubio-Palis Y. & Acevedo P. (2000). Identificación de criaderos de anofelinos en un área endémica del estado Bolívar, Venezuela. Bol. Dir. Malariol. San. Amb. 40: 21-30. [ Links ]
17.Moreno J., Rubio-Palis Y., Martínez A. & Acevedo P. (2014). Evolución espacial y temporal de la malaria en el municipio Sifontes del estado
Bolívar, Venezuela. 1980-2013. Bol. Mal. Salud Amb. 54: 236-249.
18.Moreno J., Rubio-Palis Y., Páez E., Pérez E. & Sánchez V. (2007). Abundance, biting behaviour and parous rate of anopheline mosquito species in relation to malaria incidence in gold-mining áreas of Southern Venezuela. Med. Vet. Entomol. 21: 339-349. [ Links ]
19.Moreno J., Rubio-Palis Y., Páez E., Pérez E., Sánchez V. & Vaccari E. (2009). Malaria entomological inoculation rate in gold mining areas of Southern Venezuela. Mem. Inst. Oswaldo Cruz. 104: 764-768. [ Links ]
20.Moreno J., Rubio-Palis Y., Sánchez V. & Mariany D. (2004). Primer registro de Anopheles (Nyssorhynchus) nuneztovari Gabaldon, 1940
(Diptera: Culicidae) en el estado Bolívar, Venezuela y sus implicaciones eco-epidemiológicas. Entomotrop. 19: 55-58.
21.Moreno J., Rubio-Palis Y., Sánchez V. & Martínez A. (2015a). Caracterización de hábitats larvales de anofelinos en el municipio Sifontes del estado Bolívar, Venezuela. Bol. Mal. Salud Amb. 55: 119-127. [ Links ]
22.Moreno J., Rubio-Palis Y., Sánchez V. & Martínez A. (2015b). Fluctuación poblacional y hábitat larval de anofelinos en el municipio Sifontes, estado Bolívar, Venezuela. Bol. Mal. Salud Amb.. 55: 52-68. [ Links ]
23.Moutinho P., Soares L., Cruz R. & Martins P. (2011). Population dynamics, structure and behavior of Anopheles darlingi in a rural settlement in the Amazon rainforest of Acre, Brazil. Malar. J. 10: 174. doi: 10.1186/1475-2875-10-174. [ Links ]
24.Padley D., Moody A., Chiodini P. & Saldanha J. (2003). Use of a rapid, singleround, mutiplex PCR to detect malarial parasites and identify the species present. Ann Trop. Med. Parasitol. 97: 131-137. [ Links ]
25.Pattanayak S., Dickinson K., Corey C., Murray B., Sills E. & Kramer R. (2006). Deforestation, malaria and poverty: a call for transdisciplinary research to support the design of cross-sectoral policies. SSPP2: 45-56. [ Links ]
26.Póvoa N., Lessa de Souza R., Da Luz Lacerda R., Santa Rosa E., Galiza D., De Souza J., et al. (2006). The importance of Anopheles albitarsis E and An. darlingi in human malaria transmission in Boa Vista, State of Roirama, Brazil. Mem. Inst. Oswaldo Cruz. 101: 162-168. [ Links ]
27.Rivero J., Urdaneta L., Zoghbi N., Pernalete M., Rubio-Palis Y. & Herrera F. (2004). Optimization of extraction procedure for mosquitos DNA
suitable for PCR-based techniques. Int. J. Trop. Insect. Sci. 24: 266-269.
28.Rosero D., Naranjo-Diaz N., Alvarez N., Cienfuegos A., Torres C., Luckhart S., et al. (2013). Colombian Anophelestriannulatus (Diptera: Culicidae) Naturally Infected with Plasmodium spp. ISRN Parasitol. doi: 10.5402/2013/927453. [ Links ]
29.Rubio J., Benito A., Roche J., Berzosa P., García M., Micó M., et al. (1999). Semi nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea. Am. J. Trop. Med. Hyg. 60: 183-187. [ Links ]
30.Rubio-Palis Y. (2000). Anopheles (Nyssorhynchus) de Venezuela: taxonomía, bionomía, ecología e importancia médica. Edited by: Escuela
de Malariología y Saneamiento Ambiental Dr. Arnoldo Gabaldon, Proyecto Control de Enfermedades Endémicas, Maracay.
31.Rubio-Palis Y. (2009). Prevalencia de Plasmodium spp. en anofelinos de Venezuela. Tall. ULA 12: 79-84. [ Links ]
32.Rubio-Palis Y., Ruíz-López F., Guzmán H., Sánchez V., Moreno J., Estrada Y., et al. (2013). Primer registro de Anopheles (Nyssorhynchus) oswaldoi B y Anopheles (Nys.) albitarsis F en la cuenca del río Caura, Estado Bolívar, Venezuela. Bol. Mal. Sal. Amb. 53: 68-72. [ Links ]
33.Ruíz-Lopez F., Wilkerson R., Conn J., McKeon S., Levin D., Quiñones M., et al. (2012). DNA barcoding reveals both known and novel taxa in the Albitarsis Group (Anopheles: Nyssorynchus) of Neotropical malaria vectors. Parasit. Vectors 5: 44. doi: 10.1186/1756-3305-5-44. [ Links ]
34.Sinka M., Rubio-Palis Y., Manguin S., Patil A., Temperley W., Gething P., et al. (2010). The dominant Anopheles vectors of human malaria in the Americas: occurrence data, distribution maps and bionomic précis. Parasit. Vectors. 3: 72. [ Links ]
35.Snounou G., Viriyakosol S., Zhu Z., Jara W., Pinheiro L., Rosario V., et al. (1993). High sensitivity of detection of human malaria parasites by the use of Nested Polymerase Chain Reaction. Mol. Biochem. Parasitol. 61: 315-329. [ Links ]
36.Vittor A., Pan W., Gilman R., Tielsch J., Glass G., Shields T., et al. (2009).Linking Deforestation to Malaria in the Amazon: Characterization of the Breeding Habitat of the Principal Malaria Vector, Anopheles darlingi. Am. J. Trop. Med. Hyg. 81: 5-12. [ Links ]
37.WHO (1975). Manual on practical entomology, Parts I and II. Edited by WHO, Geneva, Switzerland. [ Links ]
38.WHO (2016). World Malaria Report 2016. Edited by WHO, Geneva, Switzerland.
