¹ Cátedra de Parasitología, Escuela de Medicina "Luis Razetti" - Facultad de Medicina. Universidad Central de Venezuela, Caracas. *Autor de correspondencia: albinawide@yahoo.com

² Centro para Estudios sobre Malaria, Servicio Autónomo Instituto de Altos Estudios "Dr. Arnoldo Gabaldon", Instituto Nacional de Higiene "Rafael Rangel", Ministerio del Poder Popular para la Salud, Caracas.

³ Laboratorio de Inmunoparasitología del Centro de Microbiología y Biología Celular, IVIC, Altos de Pipe, Estado Miranda.

⁴ Departamento de Parasitología, Facultad de Farmacia, Universitat de Valencia, España.

⁵ Instituto Venezolano del Seguro Social, Hospital Domingo Luciani, Caracas, Venezuela.

⁶ Centro de Biociencias. Instituto de Estudios Avanzados (IDEA), Sartenejas, Caracas.

⁷ Sección de Biohelmintiasis-Instituto de Medicina Tropical. Facultad de Medicina. Universidad Central de Venezuela, Caracas.

⁸ Laboratorio de Biotecnología. Instituto de Medicina Tropical. Facultad de Medicina. Universidad Central de Venezuela, Caracas.

Malaria remains as a public health problem in Venezuela. In 2015 there were 136,402 cases reported by the Ministry of Popular Power for Health, being the parasite prevalence 73.95% for Plasmodium vivax, 17.6% for Plasmodium falciparum, 0.0095% for Plasmodium malariae and 8.42% mixed infections (P. vivax + P. falciparum). During the period 1999-2002 the number of cases in Venezuela ranged between 21,685 and 29,337, being the Sucre State with highest levels of malaria prevalence, with Plasmodium vivax as the unique specie in this region. In 2002 the Municipality of Cajigal had the highest Annual Parasite Incidence (API) of country, being 260 cases per 1000 inhabitants. In view of the difficulty in controlling malaria in this area, the prevalence of asymptomatic carriers was investigated as one of the epidemiological factors contributing to the persistence of malaria transmission. One hundred fifty people were included in the study, with no history of recent malaria infection, or any symptom and also, not having used antimalarial drugs during the 30 days prior to study entry. To do this, a malaria Rapid Diagnostic Test (mRDTs) was used for the determination of antigenemic (OptiMAL®) and PCR (polymerase chain reaction) in conjunction with the reference "Gold Standard", the conventional thick and thin blood smears (TTBS). It was found a prevalence of infection of 1.33% by mRDTs and TTBS and 8% by PCR which allowed the detection of 10 asymptomatic cases in addition, with a sensitivity and specificity of 100% and 93.4% respectively. The presence of asymptomatic carriers in this area reveals the difficulties that face the Malaria Control Program in the eventual elimination of this specific malaria foci. It is necessary reinforces the maintenance of the epidemiological surveillance using more sensitive diagnostic techniques, as well as to adapt the control measures based on the current findings.

Key words: Asymptomatic malaria, Plasmodium vivax, thick and thin blood smears (TTBS), mRDTs, OptiMAL^®, PCR.

Prevalencia de infecciones asintomáticas por Plasmodium vivax en el foco malárico oriental de Venezuela

RESUMEN

La malaria sigue siendo un problema de salud pública en Venezuela. Para el año 2015 el Ministerio del Poder Popular para la Salud reportó 136.402 casos, siendo la fórmula parasitaria 73,95% para Plasmodium vivax, 17,6% para Plasmodium falciparum, 0,0095 para Plasmodium malariae y 8,42% para infecciones mixtas (P. vivax + P. falciparum). Durante el período 1999-2002, el número de casos en Venezuela estuvo entre 21.685 y 29.337, siendo el Estado Sucre el que mostró los niveles más altos de prevalencia de malaria, con P. vivax como única especie en la región. En el año 2002 el Municipio Cajigal registró el Índice Parasitario Anual (IPA) más alto del país, siendo 260 casos por 1000 habitantes. En vista de las dificultades para controlar la malaria en esta área, se investigó la prevalencia de portadores asintomáticos como uno de los factores contribuyentes en la persistencia de la transmisión malárica. Ciento cincuenta personas fueron incluidas en el estudio sin historia de infección reciente por malaria o ningún síntoma, así como no haber consumido drogas antimaláricas durante los 30 días anteriores de ingresar al estudio. Para ello, se usó la prueba rápida de diagnóstico para malaria (PRDxm) para la determinación de antígenemia (OptiMAL^®) y la técnica de biología molecular basada en la Reacción en Cadena de la Polimerasa (PCR), conjuntamente con la "prueba oro," como método convencional, la Gota Gruesa y Extendido de Sangre (GGES). Se encontró una prevalencia de infección de 1,33% por GGES y por prueba rápida de diagnóstico OptiMAL^® y 8% mediante PCR. La técnica de PCR permitió la detección adicional de 10 casos asintomáticos con una sensibilidad y especificidad del 100% y 93,4% respectivamente. La presencia de portadores asintomáticos en esta área revela las dificultades que enfrenta el Programa de Control de la Malaria en la eliminación eventual de esta parasitosis en este foco. Es necesario reforzar el mantenimiento de la vigilancia epidemiológica usando técnicas de diagnóstico más sensibles, así como adoptar medidas de control basadas en estos hallazgos.

Palabras clave: Malaria asintomática, Plasmodium vivax, Gota Gruesa y Extendido de Sangre (GGES). Prueba rápida de diagnóstico OptiMAL^®, PCR.

Recibido el 18/08/2016  Aceptado el 20/12/2016

INTRODUCTION

The clinical manifestations of malaria by P. falciparum have a wide spectrum comprising asymptomatic parasitemia, clinical malaria (parasitemia with febrile episodes), and severe malaria that can lead to death (WHO, 2000; Breman, 2001; Hommel & Gilles, 2005; Noya & Ossenkoff, 2011). In contrast, P. vivax infections in nonimmune people develop symptoms of clinical malaria with recurrent febrile paroxysms, which produces temporary disability and less severe complications (Breman, 2001; Mendis et al., 2001; Sina, 2002). However, recurrent episodes induce higher morbidity and severe malaria cases by this species (Bruce et al., 2000; Mendis et al., 2001; Baird, 2013). Recently, there has been an increase in the number of studies related to severe malaria for P. vivax in endemic countries. The results of eight relatively high quality studies showed evidence on severe malaria in P. vivax infection compared with that in P. falciparum infection, identifying that the incidence of severe malaria in patients infected with P. vivax was considerable, indicating that P. vivax is a major cause of severe malaria (Nadkar et al., 2012 ; Naing et al., 2014).

In general, asymptomatic infections have been associated with acquired immunity after repeated episodes of the disease and therefore would be more frequent in areas of stable and high malaria endemicity, where adults and some children may carry parasites in asymptomatic condition for both P. malariae (Gilles, 1986) and P. falciparum (Greenwood, 1987; Greenwood et al., 1987; Achidi, 1995; Vounatsou et al., 2000; Singer et al., 2003). However, even in areas of unstable malaria, many individuals control the infection and remain asymptomatic, with very low parasitemia making it difficult for the clinical and parasitological diagnosis in P. falciparum infections (Roper et al., 1996) and P. vivax (Barbosa et al., 2014).

Latin American National Malaria Control Programs (NMCPs) are based on early diagnosis by microscopy and appropriate treatment mainly by passive case finding. The first reports of asymptomatic infections using the TTBS were conducted in Brazil (Prata et al., 1988), which recorded less prevalence than in Africa. In Venezuela malaria morbidity is caused essentially by P. vivax. Of the 136,402 cases reported in 2015, 73.95% was caused by this species in addition of 8.42% of mixed infections (P. falciparum/P. vivax) (Bol. Integral Salud Amb., 2015). The main transmission areas in Venezuela are constituted by three foci located in Southern (Bolívar and Amazonas States), Western (Táchira, Mérida, Barinas and Apure States) and North-Eastern (Sucre, Monagas, Anzoátegui and Delta Amacuro States) (Noya, 2011). The last focus was reactivated in 1985, after 20 years of being eliminated by the Malaria Control Program (Barrera et al., 1999).

It was performed a cross-sectional study based on the active search for asymptomatic malaria cases in the Cajigal Municipality of the Sucre State, located northeast of Venezuela (Fig. 1) (10° 34' 6.9'' north and 62º 49' 44'' west), with an approximate area of 365 km² and a population of 25,722 inhabitants (OCEI., 2000). In 2002, the annual parasite index (API) of the municipality was the highest in the country (260 cases per 1000 inhabitants) Cáceres (2004). After the implementation of highly effective interventions, of control program, the API decreased to 68 API/1000 inhabitants in 2003 (Cáceres, 2004). This random sampling was conducted in the first two weeks of September 2003, corresponding to the end of the rainy season and based on the area where were reported malaria cases a month earlier (Bol. Integral Salud Amb., 2003).

Individuals were notified about the characteristics of the study and agreed to participate by signing the Informed Consent Form. Adults, as well as the parents of all children between 5 to 17 years old included in the study, were asked to sign an informed consent form signed.

Two types of blood samples were collected: capillary in the earlobe for parasitological diagnosis (TTBS) and venous (OptiMAL® and PCR tests), because the anticoagulant interferes with the quality of the parasitological method and therefore with the diagnosis of the infection. In addition, urine samples were collected for determination of antimalarial drugs. Sampling was carried out based on the area where they were reported malaria cases month earlier. This research was approved and conducted under the

Parasitological diagnosis was performed by examination by thick and thin blood smears (TTBS) stained with Giemsa. Slides were examined by two independent well trained observers. At least 100 fields of the thick blood film with 100X objective and 10X eyepiece were examined before considering a negative sample (Bruce-Chwatt, 1986; Ferreira & Avila, 2001). Parasitemia was determined in the thick blood film in all positive samples (OMS, 1992; Makler et al., 1998).

Detection of parasite circulating was carried out by a malaria Rapid Diagnostic Test (mRDT) OptiMAL® (Flow Inc., Portland OR, USA) according to the manufacturer's instructions.

It was based on the amplification of specific sequences of genus and species of ribosomal genes corresponding to the 18 S subunit of RNA of the parasites for the genus Plasmodium. The total genomic DNA extraction was performed from blood collected in EDTA tubes, with the modified method of phenol-chloroform and subsequent ethanol precipitation described by Snounou et al. (1993). The DNA obtained was quantified by spectrophotometry and run in 1% (p/v) agarose gel to assess the quality of DNA.

The sensitivity of the PCR test was performed using as a source of parasites, in vitro cultures of the P. falciparum FCB2 strain (Trager & Jensen, 1976). A sample with predominance of young trophozoites, was taken to a 1% parasitemia and diluted with uninfected blood for a range of parasites from 0.1% to 0.00001%.

A nested PCR was carried out with two rounds, the first for detecting the Plasmodium and a second one specific for P. falciparum and P. vivax. (Singh et al., 1999). The primer pairs for the detection and identification of genus were rPLU1/rPLU5; rPLU3/rPLU4. The final product was analyzed and only positive samples were examined with the oligonucleotides rFAL1-rFAL2 and rVIV1-rVIV2 specific for P. falciparum and P. vivax respectively (Singh et al., 1999).

The amplified products of the second round were separated by size on 2% (p/v) agarose gels, in parallel with the appropriate molecular weight marker, stained with ethidium bromide and visualized under UV light (Singh et al., 1999).

It was used the thin layer chromatography technique described by Betschart et al. (1986), for the determination of chloroquine (CQ) and its main metabolite desethylchloroquine (DCQ) as well as quinine (QUIN), amodiaquine (AMO) and mefloquine in urine. In each run were included drug controls, untreated individuals control and monitoring of patients who had received the previous day chloroquine and primaquine. The presence of chloroquine and other antimalaric drugs was evaluated in a dark room using a UV lamp.

RESULTS

Prevalence of infection with Plasmodium spp. through thick and thin blood smears

A total of 2 asymptomatic people who represented the 1.33% (2/150) of the study group were positive for P. vivax (Table I), with densities between 440 and 1200 parasites/μL of blood. Both samples were from males, aged 11 and 20 years respectively. These people were followed up by clinical, parasitological and immunodiagnostic tests, demonstrating the persistence of parasitemia and positivity by OptiMAL® with the absence of symptoms. During follow-up, variations in parasitemia were observed in one of them and a downward trend in the other (Fig. 2, Table I). After 5 and 7 days of follow-up, patients received the conventional malarial treatment, achieving the clearance of parasites. The treatment antimalaric were based on Chloroquine 25 mg/Kg weight for 3 days distributed in 10, 10 and 5 mg/Kg weight and Primaquine (0,25 mg/Kg weight) for 14 days. It was approved and supervised for Malaria Control Program.

Prevalence of infection with Plasmodium spp. by the rapid by diagnostic test OptiMAL®

With OptiMAL^® rapid test were positive 2.0% (3/150) for Plasmodium, of which 1.33% (2/150) corresponded to P. vivax and 0.66% (1/150) to P. falciparum (Table II), the latter showing a weak positive signal. This individual was monitored by PCR and TTBS being negative for both tests. The sensitivity limit of OptiMAL^® was ≤ 300 parasites/μL of blood (Zerpa et al., 2008).

Of the 150 samples evaluated, 12 were positive for the genus Plasmodium, and subsequently tested with oligonucleotides specific for P. vivax and P. falciparum, amplifying all a product of 117 bp corresponding to P. vivax, representing a prevalence

of 8% of infection by this specie while all were negative for P. falciparum (Fig. 4).

There was concordance in the detection of 2 malaria infections by TTBS, PCR and OptiMAL^® rapid test. A positive sample for OptiMAL^® rapid test was negative by TTBS and PCR. On the other hand, ten samples were only positive by PCR. The kappa index between TTBS and OptiMAL^® rapid test was 0.796 and the kappa index between TTBS and PCR was 0.265, considering the TTBS the Gold Standard test (Table II).

Eleven of twelve people with asymptomatic infection had malaric history and another one had not malaric history, with a Mean (X) of 4.36 and a Median (X) of 4) (Table I). Two individuals with different number malaric episodes showed parasitemia levels that are over the microscopic threshold for detection remaining asymptomatic for 5 and 7 days.

Asymptomatic malaria is defined as Plasmodium infections that do not lead to clinical symptoms and therefore remain undetected by surveillance systems. These infections can still contribute to the transmission of the parasite in the population, which makes them particularly relevant in the context of malaria elimination. There are few studies about asymptomatic malaria probably due to absence of standard diagnostic criteria, since infected individuals may be in a pre-symptomatic period with parasitemia, and that could present symptoms later on (Laishram et al., 2012; Galatas et al., 2016).

In many studies the definition of asymptomatic malaria is based on the presence of parasites in peripheral TTBS, an axillary temperature <37.5ºC and the lack of malaria-related symptoms. Some studies include other criteria, such as longitudinal follow-up and parasite quantification. A longitudinal follow-up is important for differentiating between infections that seems to be asymptomatic at the moment of diagnosis, but that could become symptomatic after the initial detection (Laishram et al., 2012; Galatas et al., 2016).

There are few studies related to the pathophysiology of asymptomatic malaria infection, especially in the case of P. vivax infections. In spite of it has been proved that these individuals might influence transmission dynamics, since they might reach a prevalence 4-5 times higher than the symptomatic individuals and also, being infective for anophelines (Alves et al., 2005; Laishram et al., 2012). In the Cajigal Municipality the prevalence of asymptomatic cases was 8%. In Brazil, Alves et al. (2005) found an infection rate in An. darlingi of 1.2% for the asymptomatic carriers and 22% for the symptomatic carriers. Although the asymptomatic group infected mosquitoes at a much lower rate, these carriers usually remain infective longer than treated, symptomatic.

Also, the Cajigal Municipality is the most receptive malarious area in eastern Venezuela, reporting usually the highest prevalence in the Sucre State. Epidemiologically is considered an area of annual malaria transmission that might be responsible of exposure related immunity of the residents of this area. In fact, 11 of the 12 people with asymptomatic infection had 3-15 episodes of malaria, with a Mean (X) of 4.36 and a Median (X) of 4), which allows to infer that have developed some degree of immunity, thereby maintaining parasitemia without presenting a characteristic symptom, although not confirmed through immunological tests.

It has been well documented the presence of asymptomatic carriers in different geographic areas worldwide, that is the case of Brazil, Ethiopia and Nigeria (Prata et al.; 1988; Aranha et al., 1996; Alves et al., 2002; Barbosa et al., 2014; Tadesse et al., 2015), as well as the Amazonian focus of southern Venezuela (Postigo et al., 1998; Urdaneta et al., 2003 (unpublished data); Marcano et al., 2004), therefore it was also important to demonstrate its presence in the North-eastern focus of Venezuela (Sucre State), where it is only present P. vivax (Sojo-Milano, 2008).

In this research, it was found a prevalence of 8% of asymptomatic infections by P. vivax using PCR technique, confirming its higher sensitivity and specificity in relation to the TTBS and also, allowed to confirm that there were no P. falciparum infections. The present investigation is one of the few conducted in Venezuela focused on the detection of asymptomatic infections caused by Plasmodium species. Furthermore, this research was done in an area where there is only transmission for P. vivax and only few P. falciparum imported cases were from other endemic areas. This is one of the salient features of this study, since most of the other studies have been conducted in areas where P. falciparum was also present.

In Venezuela, there have been studies focused mainly on the Southern (Amazonas and Bolívar States) and Eastern focus (Anzoátegui, Monagas and Sucre States), where Postigo et al. (1998) reported asymptomatic malaria infections in 6% of the population that it did not detect by microscopy. In other areas of the Brazilian Amazon, asymptomatic malaria infection has been demonstrated in several cohorts of a longitudinal study, recording prevalence of 4.6, 4.2 and 0% respectively (Alves et al., 2002).

Imwong et al. (2015) found in cross-sectional surveys in western Cambodia, the Thailand–Myanmar border and southwest Vietnam, where parasite prevalence was 224/5008 (4%) by mRDTs, 229/5111 (5%) by microscopy, and 988/4975 (20%) when assessed by HVUSqPCR; of these 164 (3%) were infected with P. falciparum, 357 (7%) with P. vivax, 56 (1%) with a mixed infection, and 411 (8%) had parasite densities that were too low for species identification. In Ngella, Central Islands Province, Solomon Islands (Southwest Pacific) was detected 468 Plasmodium spp. infections (prevalence=13.4%; 463 P. vivax, five mixed P. falciparum/P. vivax, no P. ovale or P. malariae) by qPCR, whereas by light microscopy 130 positive cases (prevalence = 3.7%; 126 P. vivax, three P. falciparum and one P. falciparum/P. vivax). The prevalence of P. vivax infection varied significantly among villages (range 3.0–38.5%, p<0.001) (Waltmann et al., 2015). Although the TTBS is the common gold standard test for the diagnosis of malaria, however lacks sensitivity to detect asymptomatic infections by Plasmodium spp. (Barbosa et al., 2014).

In this research the two infections identified by the conventional method were confirmed by PCR and OptiMAL® as P. vivax, one case weakly positive for P. falciparum for OptiMAL^® rapid test was negative by PCR, so it was considered as a false positive. Since corresponded to a person who had left the Sucre State, and had never received blood transfusion and therefore unlikely P. falciparum infection. The sensitivity and specificity of OptiMAL^® rapid test in this case based on the TTBS, was 100 and 99.32% respectively. Equivalent results were

reported in a study in Thailand, where they found flaws in the diagnosis of malaria by OptiMAL® rapid test, between 25.9% and 60.3% false positives for P. vivax and for P. falciparum respectively (Coleman et al., 2002). These false positives could be explained by nonspecific binding of monoclonal antibodies conjugated to colloidal gold to capture. However, in a study carried out in Venezuela using this test it was not found inespecificity (Zerpa et al., 2008). The sensitivity and specificity of the PCR respected to the TTBS Gold Standard, was 100 and 93.24% respectively, however, the rate of concordance between these two tests was very low (κ = 0.265), which is a consequence of the greater number of samples detected by PCR, while de TTBS did not detect 10 asymptomatic carriers.

The prevalence of asymptomatic carrier was lower than those obtained previously in southern Venezuela, in mining towns (7% prevalence by TTBS and 34% prevalence by PCR) (Urdaneta et al., 2003, (unpublished data)) and 42%, 46% and 49% in indigenous populations in Amazonian State (Rodríguez et al., 2010). The PCR is a technique able to detect 1-5 parasites/μL of blood of the five species of Plasmodium that affect humans and also the possibility to diagnose mixed infections (Singh et al., 1999; Cox-Singh et al., 2007), with a sensitivity 10 times higher than the TTBS and a specificity of 100% under good laboratory practices.

In spite of the fact that the molecular technologies are costly, more time consuming and requires equipment and trained personnel., they should be included in the central laboratories of Malaria Control Program, since they allow the detection of very low parasitemias, still below the threshold of diagnosis for microscopy or rapid tests. This would allow a periodic vigilance for the search of asymptomatic infections as it has been used in other countries (Harris et al., 2010; Zoghi et al., 2012).

The Authors stated that there were no conflicts of interest in the performance of this work.

Dr. Lety González and Néstor Rubio and the Malariology staff from Yaguaraparo (Quebrada La Niña) for giving support in logistics for the field trip. Also, to Dr. Rafael Borges by helping with the statistical analysis. To the Yaguaraparo community for their cooperation in the study; Dr. Santiago Mas-Coma and José Guillermo Esteban and from the Department of Parasitology of the University of Valencia, Spain; Dr. Ananias Escalante and Ms. Esther Durrego from Laboratorio de Ecología y Genética de Poblaciones Instituto de Investigaciones Científicas-IVIC, for allowing us to do some of the PCR assays in their laboratories. Especially Dra. Hilda Pérez for the use of facilities Laboratorio de Inmunoparasitología Instituto de Investigaciones Científicas-IVIC, Altos de Pipe.

1. Achidi E. A. (1995). Asymptomatic malaria parasitaemia & seroactivities to Plasmodium falciparum in blood donors from Ibadan, south-western Nigeria. Ann. Trop. Med. Parasitol. 89: 601- 610.         [ Links ]

2. lves F. P., Durlacher R., Meneses M., Krieger H., Pereira Da Silva L. H. & Camargo E. (2002). High prevalence of asymptomatic Plasmodium vivax and Plasmodium falciparum infections in native Amazonian Populations. Am. J. Trop. Med. Hyg. 66: 641-648.         [ Links ]

3. Alves F. P, Gil L. H, Marrelli M.T, Ribolla P. E, Camargo E & Da Silva. LH. ( 2005). Asymptomatic carriers of Plasmodium spp. as infection source for malaria vector mosquitoes in the Brazilian Amazon. J. Med. Entomol. 42: 777-779.         [ Links ]

4. Aranha C. L. M., Dulccini D. G. M., Urbano F. M., De Mello Gurgel S., Escobar A. L., Marques A. et al. (1996). Hypoendemic malaria in Rondonia (Brazil, Western Amazon Region): Seasonal Variation and Risk Groups in an Urban Locality. Am. J. Trop. Med. Hyg. 55: 32-38.         [ Links ]

5. Baird K. (2013). Evidence and implications of mortality associated with acute Plasmodium vivax malaria. Clin. Microbiol. Rev. 26: 36-57.         [ Links ]

6. Barbosa S., Gozze A., Lima N., Batista C., Bastos M., Nicolete V., et al. (2014). Epidemiology of Disappearing Plasmodium vivax malaria: a case study in rural Amazonia. PLOS Negl. Trop. Dis. 8: e3109- e3122.         [ Links ]

7. Barrera R. Grillet M. E., Rangel Y., Berti J. & Aché A. (1999). Temporal and spatial patterns of malaria reinfection in northeastern Venezuela. Am. J. Trop. Med. Hyg. 61: 784- 790.         [ Links ]

8. Betschart B. & Steiger S. (1986). Quantitative determination of chloroquine and desethylchloroquine in biological fluids by high performance thin layer chromatography. Acta Trop. 43: 125-130.         [ Links ]

9. Boletín Epidemiológico Nº 52. (2003). Dirección. Salud Ambiental/ Contraloría Sanitaria. Ministerio de Salud y Desarrollo Social, Venezuela.         [ Links ]

11. Breman J. G. (2001). The ears of the hipopopotamus: Manifestations, determinants and estimates of the malaria burden. Am. J. Trop. Med. Hyg. 64(1-2 Suppl.): 1-11.         [ Links ]

12. Bruce-Chwatt L. J. (1986). Essential Malariology. First ed. Williams Heinemann Medical Books ltd. London. pp. 103-126 and 354.         [ Links ]

13. Bruce M. C., Donnelly C. A., Packer M., Lagog M., Gibson N., Narara A., et al. (2000). Age and species-specific duration of infection in asymtpomatic malaria infections in Papua New Guinea. Parasitol. 121: 247-256. William Heinemann Medical Books Ltd. London.         [ Links ]

14. Cáceres J. L. (2004). Reporte Epidemiológico Estado Sucre: El Éxito Antimalárico de Venezuela en el Año 2003. Bol. Malariol. Salud Amb. 44: 51-55.         [ Links ]

15. Coleman R. E., Maneechai N., Ponlawat A., Kumpikat C., Rachapaew N., Miller R. S., et al. (2002). Short report: Failure of the OptiMAL^® rapid malaria test as a tool for the detection of asymptomatic malaria in an area of Thail and endemic for Plasmodium falciparum and P. vivax. Am. J. Trop. Med. Hyg. 60: 563-565.         [ Links ]

16. Cox-Singh J., Davis T. M, Lee K. S., Shamsul S. S. G., Matusop A., Ratnam S., et al. (2007). Plasmodium knowlesi is widely distributed and potencially life threatening. Clin. Infec. Dis. 46: 165 - 171.         [ Links ]

17. Cucunuba Z. M., Guerra A. P., Rahirant S. J., Rivera J. A., Cortes L. J. & Nicholls R. S. (2008). Asymptomatic Plasmodium spp. Infection in Tierralta, Colombia. Mem. Inst. Oswaldo Cruz. 103: 668-673.         [ Links ]

18. Ferreira W. & Avila S. L. M. (2001). Diagnóstico Laboratorial das principais doencas infecciosa e auto-inmunes. 2ª Edicao. Editorial Guanabara Koogan . Rio de Janeiro.         [ Links ]

19. Fleiss J. L. (1981). Statistical Methods for rates and proportions. Second Edition. New York. John Wiley & Sons.         [ Links ]

20. Galatas B., Bassat Q. & Mayor A. (2016). Malaria Parasites in the Asymptomatic: Looking for the Hay in the Haystack. Trends Parasitol. 32(Issue 4): 296-308.         [ Links ]

21. Gilles H. (1986). Tropical clinical epidemiology- "A new name for an old art". Trans. R. Soc. Trop. Med. Hyg. 80: 353-359.         [ Links ]

22. Greenwood B. M. (1987). Asymptomatic malaria infections - Do They Matter?. Parasitol. Today. 3: 206-214.         [ Links ]

23. Greenwood B. M., Bradley A .K., Greenwood A. M., Byass P., Jammeh K., Marsh K., et al. (1987). Mortality and morbility from malaria among children in a rural area of the Gambia, West Africa. Trans. R. Soc. Trop. Med. Hyg. 81: 478-486.         [ Links ]

24. Harris I., Sharrock W. W., Bain L. M., Gray K. A., Bobogare A., Boaz L., et al. (2010). A large proportion of asymptomatic Plasmodium infections        [ Links ]

25. with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting. Malar. J. 9: 254.         [ Links ]

26. Hommel M. & Guilles H. M. (2005). Malaria, Tapley and Wilson’s. In: Microbiology and microbial infections. 10th edition. Volumen editors. Cox F. E., Kreeier J. P. Wakelin D. London. pp. 361-409.         [ Links ]

27. Imwong M., Nguyen T. N., Tripura R., Peto T. J., Lee S. J., Lwin K. M., et al. (2015). The epidemiology of subclinical malaria infections in South-East Asia: findings from cross-sectional surveys in Thailand–Myanmar border areas, Cambodia, and Vietnam. Malar. J. 14: 381.         [ Links ]

28. Laishram D., Sutton P., Nanda N., Sharma J., Sobti R., Carlton J., et al. (2012). The complexities of malaria disease manifestations with a focus on asymptomatic malaria. Malar. J. 11: 29-44.         [ Links ]

29. Makler M. T., Palmer C. J. & Ager A. L. (1998). A review of practical techniques for the diagnosis of malaria. Ann. Trop. Med. Parasitol. 92: 419-433.         [ Links ]

30. Marcano T. J., Morgado A., Tosta C. E. & Coura J. R. (2004). Cross-sectional study defines difference in malaria morbidity in two Yanomami communities on Amazonian boundary between Brazil and Venezuela. Mem. Inst. Oswaldo Cruz. 99: 369-376.         [ Links ]

31. Mendis K., Sina B., Marchesini P. & Carter R. (2001). The neglected burden of Plasmodium vivax malaria. Am. J. Trop. Med. Hyg. 64: 97-106.         [ Links ]

32. Nadkar M. Y., Huchche A. M., Singh R. & Pazare A. R. (2012). Clinical profile of severe Plasmodium vivax malaria in a tertiary care centre in Mumbai from June 2010–January 2011. J. Assoc. Physicians India. 60: 11–13.         [ Links ]

33. Naing C., Whittaker M. A., Nyunt Wai V. & Mak J. W. (2014). Is Plasmodium vivax Malaria a Severe Malaria? A Systematic Review and Meta-Analysis. PLOS Negl. Trop. Dis. 8(8): e3071. doi: 10.1371/journal.pntd.0003071.         [ Links ]

34. Noya O. (2011). Introducción. En: Fundamentos en el Diagnóstico y Control de la Malaria. Instituto de Altos Estudios Dr "Arnoldo Gabaldon", Ministerio del Poder Popular para la Salud, República Bolivariana de Venezuela." Maracay. pp.11-21.         [ Links ]

35. Noya O. & Ossenkop J. (2011). Diagnóstico clínico de la Malaria. En: Fundamentos en el Diagnóstico y Control de la Malaria. Instituto de Altos Estudios Dr. "Arnoldo Gabaldon", Ministerio del Poder Popular para la Salud, República Bolivariana de Venezuela." Maracay. pp.117-135.         [ Links ]

36. OCEI (2000). Estimaciones y Proyecciones de Población 1959-2025. Oficina Central de Estadística e Informática, Diciembre 1985. Caracas-Venezuela.         [ Links ]

37. OMS (1992). Métodos básicos de laboratorio en Parasitología Médica. Manual de Laboratorio. España. pp. 40-49, 83-94.         [ Links ]

38. Postigo M., Mendoza-León A. & Pérez H. (1998). Malaria diagnosis by de Polymerase chain reaction: a field study in south-eastern Venezuela. Trans. R. Soc. Trop. Med. Hyg. 92: 509-511.         [ Links ]

39. Prata A., Urdaneta M., McGreevy P. B. & Tada M. S. (1988). Infrequency asymptomatic malaria in an endemic area in Amazonas. Brazil. Rev. Soc. Bras. Med. Trop. 21: 51-54.         [ Links ]

40. Rodrigues C. J., Suárez-Mutis M. & Ladeia-Andrade S. (2006). A new challenge for malaria control in Brazil: asymptomatic Plasmodium infection – A Review. Mem. Inst. Oswaldo Cruz. 101: 229-237.         [ Links ]

41. Rodríguez I., De Abreu N., Carrasquel A., Bolívar J., González M., Scorza J. V., et al. (2010). Infecciones maláricas en individuos asintomáticos en la población indígena Jivi, Amazonas, Venezuela. Bol. Mal. Sal. Amb. 50: 198-205.         [ Links ]

42. Rodulfo H., De Donato M., Mora R., González L. & Contreras C. E. (2007). Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela. Brazilian J. Med. Biol. Res. 40: 535-543.         [ Links ]

43. Roper C., Elhassan I. M., Hviid l., Giha H., Richardson W., Babiker H., et al. (1996). Detection of very low level Plasmodium falciparum Infections using the nested polymerase chain reaction and reassessment of the epidemiology of unstable Malaria in Sudan. Am. J. Trop. Med. Hyg. 54: 325-331.         [ Links ]

44. Sina B. (2002). Focus on Plasmodium vivax. Trends Parasitol. 18: 287–289.         [ Links ]

45. Singer L. M., Mirel L. B., Ter Kuile F. O., Branch O. H., Vulule J. M., Kolczak M., et al. (2003). The effects of varying exposure to malaria transmission on development of antimalarial antibody responses in preschool children XVI, AsemboBay Cohort Project. J. Infect. Dis. 187: 1756-1764.         [ Links ]

46. Singh B., Bobogare A., Cox-Singh J., Snounou G., Shukri M. & Rahman A. (1999). A genus and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am. J. Trop. Med. Hyg. 60: 687-692.         [ Links ]

47. Snounou G., Viriyakosol S., Jarra W., Thaithong S. & Brown K. N. (1993). Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol. Biochem. Parasitol. 58: 283-293.         [ Links ]

48. Sojo-Milano M., Cáceres J., Pizzo N., Rojas J., Maldonado A., Rubio-Pulgar N., et al. (2008). Malaria recurrente a Plasmodium vivax. Municipio Cajigal, Estado Sucre, Venezuela. Rev. Biomed. 19: 3-15.         [ Links ]

49. Suárez-Mutis M., Cuervo P., Leoratti F. M. S., Ferreira A. W., Fernandes O. & Rodrigues Coura J. (2007). Cross sectional study reveals a high percentage of asymptomatic Plasmodium vivax infection in the Amazon Rio Negro area Brazil. Rev. Inst. Med. Trop. S. Paulo. 49: 159-164.         [ Links ]

50. Tadesse F. G., Pett H., Baidjoe A., Lanke K., Grignard L., Sutherland C., et al. (2015). Submicroscopic carriage of Plasmodium falciparum and Plasmodium vivax in a low endemic area in Ethiopia where no parasitaemia was detected by microscopy or rapid diagnostic test Malar. J. 14: 303.         [ Links ]

51. Trager W. & Jensen J. B. (1976). Human malaria parasites in continuous culture. Sci. 193: 125-129.         [ Links ]

52. Vallejo A. F., Chaparro P. E., Benavides Y., Álvarez A., Quintero J. P., Padilla J., et al. (2015). High prevalence of sub-microscopic infections in Colombia. Malar. J. 14: 201.         [ Links ]

53. Vounatsou P., Smith T., Kitua A. Y., Alonso P. & Tanner M. (2000). Apparent tolerance of Plasmodium falciparum in infants in a highly endemic area. Parasitol. 120: 1-9.         [ Links ]

54. Waltmann A., Darcy A. W., Harris I., Koepfli C., Lodo J, Vahi V., et al. (2015). High Rates of Asymptomatic, Submicroscopic Plasmodium vivax Infection and Disappearing Plasmodium falciparum Malaria in an Area of Low Transmission in Solomon Islands. PLOS Negl. Trop. Dis. 9(5): e0003758. doi:10.1371/journal.pntd.0003758.         [ Links ]

55. WHO (2000). Severe and complicated malaria. Trans. R. Soc. Trop. Med. Hyg. 94(suppl): 51-90.         [ Links ]

56. Zerpa N., Pabón R., Wide A., Gavidia M., Medina M., Cáceres J. L., et al. (2008). Evaluation of the OptiMAL^® test for diagnosis of malaria in Venezuela. Invest. Clín. 49: 93 -101.         [ Links ]

57. Zoghi S., Mehrizi A. A., Raeisi A., Haghdoost A. A., Turki H., Safari R., et al. (2012). Survey for asymptomatic malaria cases in low transmission settings of Iran under elimination programme. Malaria J. 11: 126.         [ Links ]