Meta, Colombia
[Paper written according to guidelines for Medical and Veterinary Entomology]
Sebastián Durán
1, Luz Stella Buitrago
2, Jan E. Conn
3,4, Helena
Brochero
1*1
Facultad de Agronomía, Universidad Nacional de Colombia, Bogotá DC, Colombia 2
Unidad de Entomología, Secretaría Departamental de Salud del Meta, Villavicencio, Meta, Colombia
3
Griffin Laboratory, Wadsworth Center, New York State Department of Health, Slingerlands, NY, 12159, USA
4
Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY, 12222, USA
*Corresponding author
ABSTRACT
Previous data demonstrate urban malaria transmission in Puerto Gaitán (Meta, Colombia), a large municipality located in the Orinoquia region. This 9 month-long study, carried out on 2009, used human landing catch (HLC, between 18H and 06H) to obtain anopheline specimens. An. darlingi is considered to be the main malaria vector in Meta Department, and during this study it was the most prevalent species (n=3116) and exhibited biting activity at dusk and dawn, with three peak biting times indoor (21-22h, 23-24h and 2-3h); and two outdoor (21-22h and 2-3h).
An. braziliensis (n=412) bit throughout the night, both indoor and outdoor, and was
collected from the onset of the rainy season to the end of the sampling period (May-Dec). An. albitarsis s.l. was collected only during the rainy season (Jul-Dec), its biting activity indoor peaked at dusk and from 02 to 03h; whereas its activity outdoor was high at dusk, dawn and showed peaks from 00 to 01h and from 02 to 03h. Other species collected were An. oswaldoi s.l. (n=2), An. nuneztovari (n=4),
An argyritarsis (n=3) and An. peryassui (n=4). Only one specimen, An. albitarsis F
of the Albitarsis Complex, identified as such with the COI barcode, was found infected with P. falciparum. Interestingly, the infected An. albitarsis F was collected in a neighborhood with high malaria incidence. Despite being the most abundant species, An. darlingi was not infected with any human malaria parasite. It is recommended that the local residents wear repellent and use LITN´s to reduce human-vector contact.
Capítulo 2 43
Keywords Anopheles malaria biology vector Colombia Orinoquia
INTRODUCTION
Puerto Gaitán (Meta Department), the fourth largest municipality (17424km2) in Colombia, is located in the Orinoquia region. With an average altitude of 149 m, it is mostly savannah, transformed by urban growth and economical activities such as livestock, trade, fishing, logging, African oil palm, corn, soy and oil exploitation (Cicery, Gualtero, & Cicery, 2005). It is located on the Manacacias watershed, a river that periodically floods some local urban and periurban areas (Cicery, Gualtero, & Cicery, 2005). It exhibits an average temperature of 26ºC, an annual rainfall of 2200mm, and a monomodal rainfall distribution (rainy season from March to November). Recently Puerto Gaitán has been experiencing rapid urban growth and increased population because of a newly developed oil industry.
25 Anopheles species are reported for the Meta Department (Ahumada M. , 2009), among them the main Colombian Orinoquia vector, An. darlingi (Servicio de Erradicacion de la Malaria, 1957; Jiménez, Conn, Wirtz, & Brochero, 2012). This species is also the main malaria vector in the Meta Department (Ahumada M. , 2009; Ahumada, Pareja, Buitrago, & Quiñones, 2013; Olano, Brochero, Sáenz, Quiñones, & Molina, 2001), a highly antropophilic species with endophagic and exophagic behavior (Rubio-Palis, 2000; Montoya-Lerma, et al., 2011). It has been found in forest environments and near jungles, but it also has been found in semiurban environments such as the periurban area of Quibdó (Chocó, Colombia), a locality with urban malaria transmission (Montoya-Lerma, et al., 2011; Ochoa & Osorio, 2006). Another species, An. marajoara s.s., was reported infected with P. falciparum in Puerto Carreño (Vichada, Colombia), also in the Orinoquia region, where it may be involved in malaria transmission (Jiménez, Conn, Wirtz, & Brochero, 2012). Additionally, Puerto Gaitán has been characterized as a savannah-sub region in the llanos ecoregion (Rubio-Palis & Zimmerman, 1997).
Since 2005, there is evidence of urban malaria transmission in this municipality (Buitrago, Brochero, McKeon, Lainhart, & Conn, 2013). Together, unplanned human migration and urbanization, land use changes, forced displacement caused by nearby armed conflict (Buitrago, Brochero, McKeon, Lainhart, & Conn, 2013) and location adjacent to the Departments of Vichada and Casanare, both with high malaria incidence (SIVIGILA, 2009), and are likely shape local malaria transmission dynamics (Buitrago, Brochero, McKeon, Lainhart, & Conn, 2013), and could alter Anopheles spp. biology and/or ecology.
This study aimed to 1) identify the human biting species of the genus Anopheles in the municipality of Puerto Gaitán; and 2) assess Anopheles spp. biological aspects that could account for their importance as local vectors.
44 Determinación del papel en la transmisión de malaria de las especies de Anopheles
MATERIALS AND METHODS
Study area
This study was performed in Puerto Gaitán-Meta, Colombia (04°19′N, 72°05′W), during 2009 (March-May, July-December). Sampling was conducted in houses in 2 neighborhoods identified as malaria hotspots by local health authorities personnel: Paraíso Natural, Trampolín and also in Centro.
Figura 2.2-1. Puerto Gaitán (Meta, Colombia) (Wikimedia Commons)
Mosquito collections
Collections in Trampolín, Paraíso Natural and Centro were conducted for 2 consecutive nights (18H to 06H) per sampled month in Puerto Gaitán, during the new moon lunar phase or close to it (Silver & Service, 2008). Mosquitoes were collected by human landing catch (HLC) and sampling hours were divided into 50 minutes for collection and 10 minutes for notes, resting and labeling mosquito collection containers. To avoid bait bias, HLC collectors rotated each hour between indoor (main room, halls or living/dining room) and outdoor (within 10 m of the house) (WHO, 1975). Temperature (ºC) and relative humidity (%) were measured hourly. Human biting rate (per hour and per night) was calculated after taxonomic identification, and was defined as the amount of bites per person per night (Jiménez, Conn, Wirtz, & Brochero, 2012).
Capítulo 2 45
Taxonomic identification of the entomological material was carried out using dichotomous keys (Faran M. , 1980; Faran & Linthicum, 1981; González & Carrejo, 2009; Rubio-Palis, 2000). To confirm taxonomic identification, isofamilies were obtained from collected females. An isofamily is defined as a set of eggs, 4th instar larval molt, pupae molt and adult forms obtained from a single egg. To obtain isofamilies, Anopheles spp. females were taken alive to the Laboratorio de Genética de Insectos´ rearing room at Universidad Nacional de Colombia (Bogotá DC, Colombia) and blood-fed on Mus musculus. Blood-fed and unfed wild-females were subjected to forced oviposition wherein each female was anesthetized using Ethyl-acetate and one wing and hind leg were removed to induce oviposition. The individual was then set on the water surface in a small cup half filled with tap water to lay eggs (Estrada, et al., 2003). Larvae were fed with ground dog food, and molts of fourth instar larvae and pupae were stored in plastic vials with 70%V/V ethanol, and adult individuals were kept in plastic 1.5ml eppendorf tubes at -20ºC.
Plasmodium spp. natural infection
Individuals were tested by ELISA (Enzyme-Linked ImmunoSorbent Assay) for circumsporozoite protein of P. falciparum, P. vivax-VK210 and P. vivax-VK247 (Wirtz R. , Burkot, Andre, Rosenberg, Collins, & Roberts, 1985; Wirtz R. , Burkot, Graves, & Andre, 1987; Wirtz R. , 2009); using the kit (antibodies and positive controls) provided by the Center for Disease Control (CDC, Atlanta, USA). Individuals (thorax and head) were pooled (same species individuals, same time, date and site of collection) for the first round of ELISA, and the cut-off value (absorbance above which a test is considered positive), for each plate, was defined as twice the mean absorbance of the negative controls (Wirtz R. , Burkot, Graves, & Andre, 1987). To confirm infection, a second round of ELISA was carried out using the remaining lysate of pools that were initially positive.
Infection was additionally confirmed via polymerase chain reaction carried using species-specific primers (Singh, Bobogare, Cox-Singh, Snounou, Abdullah, & Rahman, 1999). PCR and product visualization (electrophoresis on 2% agarose gel with ethidium bromide stain) were performed with the aid of Instituto Nacional de Salud´s entomology laboratory staff.
Taxonomic identification confirmation via molecular tools
Confirmation of taxonomic identification of individuals from positive ELISA pools was carried out via sequencing the barcode region of COI mtDNA and assessing these sequences using BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi). Genomic DNA was extracted from individual abdomens using QIAGEN® DNeasy Blood & Tissue kit. PCR was carried out using the using the LCO1490 and HCO2198 primers (Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994), following the next conditions (95°C for 5:00 min. 35 cycles of; 95°C for 0:30 sec, 48°C for 0:30 sec, 72°C for 0:45 sec). Sequencing
46 Determinación del papel en la transmisión de malaria de las especies de Anopheles
of the forward band was carried out by The Applied Genomics Technology Core (Wadsworth Center, USA). Sequences were edited and trimmed using Sequencher 3.0 (Gene Codes Corps, USA).
Entomological data analyses
HBR for each species was calculated as the number of female Anopheles spp. bites per person per night (Girod, Gaborit, Carinci, Issaly, & Fouque, 2008). Biting activity was visualized for each hour (Jiménez, Conn, Wirtz, & Brochero, 2012). CSP index was calculated as the proportion of mosquitoes positive for circumsporozoite protein of any of the assayed parasite species (Girod, Gaborit, Carinci, Issaly, & Fouque, 2008). EIR (Entomological Inoculation Rate) was calculated as the amount of infective bites that may be received by a person in a year, EIR=(bites/person/night)*365nights*CSP (MacDonald, 1957). Annual parasite index (API) was calculated for P. falciparum and P. vivax as follows API = (confirmed cases during 1 year/population under surveillance) x 1000.
Ethics
HLC collection protocol (#02-028a) was approved by the Institutional Review Board (IRB) of the New York State Department of Health (NYSDOH) and Universidad Nacional de Colombia; collectors signed an informed consent form.
Anopheles spp female blood feeding with Mus musculus was undertaken following
the vertebrate handling protocols (Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional de Colombia), National Institute of Health (Assurance #A5791-01) and the New York State Department of Health IACUC protocol #11- 420.
RESULTS
Mosquito collection and taxonomic identification
412 hours (206H outdoor, 206H indoor) of HLC were carried out and a total of 3732 Anopheles spp. females were identified as An. darlingi (n=3116), An.
braziliensis (n=412), An. albitarsis s.l (n=106), An. oswaldoi s.l (n=2), An. nuneztovari (n=4), An argyritarsis (n=3) and An. peryassui (n=4).
Capítulo 2 47
Figura 2.2- 2. Anopheles spp. HLC sampling points on Puerto Gaitán (Meta, Colombia)-2009 (IDEAM, WMS services), see Tabla 2.2-1. for additional information.
HLC nighttime collections were distributed among 2009 (Puerto Gaitán) as follows;
Neighborhood Abbreviation Sampled periods Coordenadas
Centro CNA March (1 incomplete night, 18- 24H)*
04° 18.715'N, 72° 04.680'O
Trampolín VT March (1 incomplete night, 18- 22H)*
04° 18.459'N, 72° 04.852'O
Trampolín CDM
March (1 incomplete night, 18- 22H)*
April (2 nights, 18-06H) May (2 nights, 18-06H)
04° 18.436'N, 72° 04.967'O
Paraíso Natural OVL
July (2 nights, 18-06H) August (2 nights, 18-06H) September (2 nights, 18-06H) October (2 nights, 18-06H) November (2 nights, 18-06H) December (2 nights, 18-06H) 04° 19.293'N, 72° 04.840'O
Tabla 2.2- 1. Anopheles spp. human landing catches on Puerto Gaitán-Meta (Colombia, 2009).*Not taken in count for final analyses and index calculations
Low mosquito densities on March´s (2009) nights caused early (22H, 24H) HLC suspensions (Table 2.2-1). An. darlingi mean HBR was 170.0, mean HBR of 27.5 for An. braziliensis and 6.1 for An. albitarsis sl.
48 Determinación del papel en la transmisión de malaria de las especies de Anopheles
Figura 2.2- 3. A-An. darlingi,B- An. albitarsis sl and C- An. braziliensis, hourly biting activity on Puerto Gaitán – Meta (Colombia, 2009). *March captures were not taken in count for final analyses
Capítulo 2 49
Figura 2.2- 4. An. darlingi, An. braziliensis and An. albitarsis sl monthly nighttime biting activity on Puerto Gaitán – Meta (Colombia, 2009). A. Indoor, B. Outdoor. *March captures were not taken in
count for final analyses
Plasmodium spp. natural infection
A pool with 2 individuals originally identified as An. braziliensis (outdoor; 23-24H; September 16, 2009; Paraíso Natural -OVL-) was positive for P. falciparum for both ELISA rounds. After DNA extraction from individual abdomens using QIAGEN® DNeasy Blood & Tissue kit, both extracts were subjected for PCR to amplify the barcode region from COI mtDNA (Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994) to verify the initial mosquito identification. COI sequences were assessed using BLAST (Altschul, Gish, Miller, Myers, & Lipman, 1990) and BOLD (Ratnasingham & Hebert, 2007) search tools. We found that one of the individuals initially identified as An. braziliensis was a member of the Albitarsis complex of
Anopheles, with a high identity with An. albitarsis F sequences. A BLAST query of
50 Determinación del papel en la transmisión de malaria de las especies de Anopheles
resulted in 99.85% identity with An. albitarsis F from Cojedes (Venezuela), GenBank Accession JQ615033 (Ruiz, et al., 2012).
To determine which species (An. albitarsis F or An. braziliensis) was infected with
P. falciparum, PCR using specific primers for this parasite species (rFAL1 and
rFAL2) was carried out with the expectation of a positive reaction (Singh, Bobogare, Cox-Singh, Snounou, Abdullah, & Rahman, 1999). An. albitarsis F was found infected with P. falciparum (Figure 2.2-5), thus its CSP was 0.943% (1/106) and its EIR was 21.13 (EIR=6.14*365*0.00943). The infected individual was caught in the Paraíso natural neighborhood (OVL), at 23-24 h outdoor September 16th (2009).
Figura 2.2- 5. PCR (rFAL1-rFAL2) gel electrophoresis; 1)100 bp DNA ladder; 2) Negative PCR control; 3) Positive PCR control (P. falciparum 3D7 strain); 4) An. braziliensis; 5) An. albitarsis F; 6) 100 bp
DNA ladder.
Entomological data analyses
Because HLC was carried out in the urban area, the 127 urban malaria cases reported for 2009 (Buitrago, Brochero, McKeon, Lainhart, & Conn, 2013) were considered for the current analysis. 99 of these cases were P. vivax and 28 were
P. falciparum. Annual Parasite Index (API) of 3.966 for P. falciparum and 14.023
for P. vivax in the urban area were calculated based on the urban population of 7060 inhabitants in the 2005 national census (DANE, 2010). Most urban P. vivax cases were registered during the 2009 dry season, whereas P. falciparum cases seemed to occur during the rainy season.
DISCUSSION
The highest HBR for An. darlingi was near the end of the rainy season. An increase in crepuscular biting activity throughout the night was observed but it is not as marked as the one observed at Villavicencio-Meta (Brochero, Rey, Buitrago, & Olano, 2005; Ahumada, Pareja, Buitrago, & Quiñones, 2013). In other
Capítulo 2 51
Orinoquia region localities such as Puerto Carreño-Vichada An. darlingi was found infected by P. vivax-VK210 and showed an activity peak outdoor at 21-22 h (Puerto Gaitán, Meta) and an activity peak at 5-6 h; indoor activity peaked at 21- 22 h and 4-5 h (Jiménez, Conn, Wirtz, & Brochero, 2012). These differences in peak biting times underscore the variability in this behavior, perhaps linked to the availability of humans for bloodmeals.
For An. albitarsis s.l, the highest HBR was recorded near the end of the rainy season. Biting activities in other Orinoquia regions for this species present some variation (Fig. 2-2-3 B), with the same high activity at dusk in Puerto Carreño during the same year (Jiménez, Conn, Wirtz, & Brochero, 2012). Other activity peaks were shared with Villavicencio (Ahumada M. , 2009). Other species collected in Puerto Gaitán have been reported previously for Meta, for example, An. braziliensis, An. oswaldoi s.l. (n=2), An argyritarsis (n=3) and An. peryassui (n=4). Although elsewhere in Colombia,
An. nuneztovari is a vector, in Meta Department in this report, it has a very limited
presence (n=4) (Olano, Brochero, Sáenz, Quiñones, & Molina, 2001; Gutiérrez, y otros, 2009).
Seasonal variation of HBR could not been fully assessed due to under-sampling of the dry season, with only two months (March being incompletely sampled, December) sampled for this season, which normally goes from December to March. Although An.
darlingi and An. albitarsis HBRs were higher on the rainy season, these abundances
were not correlated with malaria case reporting throughout 2009. API values for P.
falciparum and P. vivax might actually be lower than reported here due to an
underestimation of Puerto Gaitán´s population, likely larger in 2009 than reported in 2005.
The neighborhood of Paraíso Natural, where the single P. falciparum infected An.
albitarsis F individual was caught, is one of the highest P. falciparum incidence
neighborhoods, suggesting that this species could be an important local urban vector. The An. albitarsis F EIR could be an overestimation because of incomplete sampling throughout the dry season, when it is expected to be less abundant. Additionally, the lack of sampling during January and February, when most P. vivax cases were reported, could explain lack of infected Anopheles spp. individuals detected. It is important to evaluate An. albitarsis F as vector because this is the first report of its natural infection in the Orinoquia region since it was first recorded in Puerto Carreño, Vichada (Brochero, Li, & Wilkerson, 2007).
Assuming the trend of rapid immigration continues in Puerto Gaitán, this evidence of urban malaria transmission suggests that inhabitants should focus on personal protection, such as repellents and long lasting insecticide treated nets, to reduce human-vector contact (WHO, 2013).
ACKNOWLEDGEMENTS
Secretaría Seccional de Salud del Meta (Colombia). Secretaria de Salud de Puerto Gaitán, Meta (Colombia). Labmates; Pilar Jiménez, Humberto Mosquera, Jimena Molano, Diana Ortiz and Liliana García. Martha Ahumada, Martha Quiñones,
52 Determinación del papel en la transmisión de malaria de las especies de Anopheles
Elizabeth Ruiz and the Puerto Gaitán community. Funding sources; Universidad Nacional de Colombia (QuiPu 201010012197), USA NIH AI 2R01AIO54139.
REFERENCES
Ahumada, M. (2009). Determinación del papel como vector de malaria de las especies de Anopheles presentes en el departamento del Meta, Colombia. Universidad Nacional de Colombia, Facultad de Medicina, Infecciones y Salud en el Trópico. 73.
Ahumada, M., Pareja, P., Buitrago, L., & Quiñones, M. (2013). Comportamiento de picadura de Anopheles darlingi Root, 1926 (Diptera: Culicidae) y su asociación con la transmisión de malaria en Villavicencio (Meta, Colombia). Biomédica 2013; 33(2) . Altschul, S., Gish, W., Miller, W., Myers, E., & Lipman, D. (1990). Basic local alignment search tool. J. Mol. Biol. 215:403-410.
Brasil, P., de Pina Costa, A., Pedro, R., da Silveira Bressan, C., da Silva, S., Tauil, P., et al. (2011). Unexpectedly long incubation period of Plasmodium vivax malaria, in the absence of chemoprophylaxis, in patients diagnosed outside the transmission area in Brazil. Malar J 2011 ;10:122 .
Brochero, H., Li, C., & Wilkerson, R. (2007). A newly recognized species in the Anopheles (Nyssorhynchus) albitarsis complex (Diptera: Culicidae) from Puerto Carreño, Colombia. Am J Trop Med Hyg 2007;76(6):1113-7 .
Brochero, H., Rey, G., Buitrago, L., & Olano, V. (2005). Biting activity and breeding sites of Anopheles species in the municipality Villavicencio, Meta, Colombia. J Am
Mosq Control Assoc 2005;21(2):182-6 .
Buitrago, L., Brochero, H., McKeon, S., Lainhart, W., & Conn, J. (2013). First published record of urban malaria in Puerto Gaitán, Meta, Colombia. Mem Inst
Oswaldo Cruz 108(8):1045-1050 .
Cicery, E. (2005). Monografía del municipio de Puerto Gaitán; visión geohistórica y cultural. 105pp.
DANE. (2010). Censo general 2005, Perfil Puerto Gaitán. 1-6.
Estrada, D., Quiñones, M., Sierra, D., Calle, D., Ruiz, F., Erazo, H., et al. (2003). Utilidad de la morfología de los huevos como método indirecto para identificar Anopheles benarrochi Gabaldón,Cova García & López, Anopheles oswaldoi (Peryassu) y Anopheles rangeli Gabaldón, Cova García & López, (Diptera:Culicidae) en Putumayo, Colombia. Biomédica 2003;23:388-95.
Capítulo 2 53
Faran, M. (1980). A revisión of the Albimanus section of the subgenus Nyssorhynchus. Contrib Am Entomol Inst. 1980; 15:1-215. .
Faran, M., & Linthicum, K. (1981). A handbook of the Amazonian species of Anopheles (Nyssorhynchus) (Diptera: Culicidae). Mosq Syst. 1981; 13:1-81.
Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial Cytochrome c Oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 1994; 3: 294-299 .
Girod, R., Gaborit, P., Carinci, R., Issaly, J., & Fouque, F. (2008). Anopheles darlingi bionomics and transmission of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae in Amerindian villages of the Upper-Maroni Amazonian forest, French Guiana. Mem. Inst. Oswaldo Cruz 103; 7: 702-710 .
González, R., & Carrejo, N. (2009). Introducción al estudio taxonómico de Anopheles de Colombia: claves y notas de distribución. 2da edición. Programa editorial Univalle. 260pp.
Jiménez, P., Conn, J., Wirtz, R., & Brochero, H. (2012). Anopheles (Díptera: Culicidae) vectores de malaria en el municipio de Puerto Carreño, Vichada, Colombia. Biomédica 2012;32(Supl.):13-21.
MacDonald, G. (1957). The epidemiology and control of malaria. Oxford: Oxford University Press.
Montoya-Lerma, J., Solarte, Y., Giraldo-Calderón, G., Quiñones, M., Ruiz-López, F., Wilkerson, R., et al. (2011). Malaria vector species in Colombia - A review. Mem Inst Oswaldo Cruz. 2011 Aug;106 Suppl 1:223-38.
Ochoa, J., & Osorio, L. (2006). Epidemiología de malaria urbana en Quibdó, Chocó. Biomédica 2006;26:278-85.
Olano, V., Brochero, H., Sáenz, R., Quiñones, M., & Molina, J. (2001). Mapas preliminares de la distribución de especies de Anopheles vectores de malaria en Colombia. Biomédica 2001;21:402-8.
Ratnasingham, S., & Hebert, P. (2007). bold: The Barcode of Life Data System (http://www.barcodinglife.org). Mol Ecol Notes 1;7(3):355–364 .
Rubio-Palis, Y. (2000). Anopheles (Nyssorhynchus) de Venezuela: taxonomía, bionomía, ecología e importancia médica. Escuela de Malariologia y Saneamiento Ambiental. Maracay, Venezuela. 2000; 120p.
Rubio-Palis, Y., & Zimmerman, R. (1997). Ecoregional classification of malaria vectors in the neotropics. J Med Entomol. 1997 Sep;34(5):499-510.
54 Determinación del papel en la transmisión de malaria de las especies de Anopheles
Ruiz, 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: Nyssorhynchus) of Neotropical malaria vectors. Parasites and Vectors
5(44):1-12 .
Servicio de Erradicacion de la Malaria. (1957). Plan de Erradicación de la Malaria en Colombia. Volumen I y II. Bogotá: Ministerio de Salud Nacional.
Singh, B., Bobogare, A., Cox-Singh, J., Snounou, G., Abdullah, M., & Rahman, H. (1999). A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg 60(4):687-92 .
The Walter Reed Biosystematics Unit. (n.d.). MEDICALLY IMPORTANT
MOSQUITOES. Retrieved January 20, 2014, from Southcom:
http://www.wrbu.org/SpeciesPages_ANO/ANO_A-hab/ANbra_hab.html
Warrell, D., & Gilles, H. (2002). Essential Malariology. 4th edition. New York. Oxford University Press. 2002:192.
WHO. (1975). Manual on Practical Entomology in Malaria. Part II: Methods and Techniques, Geneva, 191 pp.
WHO. (2013). World Malaria Report 2013. Geneva, Switzerland: WHO. pp284.
Wirtz, R. (2009). Sporozoite ELISA directions. Center for Disease Control and Prevention, Atlanta, USA.
Wirtz, R., Burkot, T., Andre, R., Rosenberg, R., Collins, W., & Roberts, D. (1985). Identification of Plasmodium vivax sporozoites in mosquitoes using an enzyme-linked immunosorbent assay. Am Jour Trop Med Hyg 1985; 34: 1048-1054p .
Wirtz, R., Burkot, T., Graves, P., & Andre, R. (1987). Field evaluation of enzyme-