Differential NRAMP1gene's D543N genotype frequency: Increased risk of contracting tuberculosis among Venezuelan populations.

NRAMP1 and VDR gene polymorphisms have been variably associated with susceptibility to tuberculosis (TB) amongst populations having different genetic background. NRAMP1 and VDR gene variants' association with susceptibility to active infection by Mycobacterium tuberculosis (Mtb) was analyzed in the Warao Amerindian population, an ethnic population from Venezuela's Orinoco delta region. Genomic DNA was extracted from individuals with and without TB to evaluate genetic polymorphism by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Four NRAMP1 gene polymorphisms were analyzed: D543N (rs17235409), 3' UTR (rs17235416), INT4 (rs3731865), and 274C/T (rs2276631), and one VDR gene polymorphism: FokI (rs2228570). The results showed that the genotypes D543N-A/A, 3'UTR-TGTG+/+, INT4-C/C, and 274C/T-T/T of known polymorphism in the NRAMP1 gene, as well as the genotypes FokI-F/f and FokI-f/f in the VDR gene were most often found in indigenous Warao with active TB. Binomial logistic regression was used for evaluating associations between polymorphisms and risk of contracting TB, an association between NRAMP1-D543N-A/A genotype distribution and TB susceptibility was found in Warao Amerindians. Regarding Venezuelan populations having different genetic backgrounds; statistically significant TB associations concerning NRAMP1-D543N-A/A, INT4-C/C and 3'UTR-TGTG+/+ variant genotype distributions in Warao Amerindians (indigenous) compared to Creole (admixed non-indigenous population) individuals were found. In conclusion, the results thus indicated that the association between NRAMP1-D543N-A/A genotype and TB in Warao Amerindians could support such allele's role in host susceptibility to Mtb infection.

Conocimiento, actitudes y prácticas sobre la Malaria en comunidades de Maniapure, Estado Bolívar

Introducción: La Malaria es una enfermedad de importancia epidemiológica, la cual ha tenido un repunte en los últimos años en el Estado Bolívar. Al ser potencialmente mortal, es vital la elaboración de estrategias preventivas basadas en los conocimientos y prácticas comunitarias. Objetivos: Identificar los conocimientos, actitudes y prácticas relacionados con la malaria y comparar con hallazgos de otras áreas endémicas. Métodos: Se realizó un estudio observacional, descriptivo y de corte transversal en la región de Maniapure del 28 al 31 de agosto del 2018. Se aplicó un cuestionario de 30 preguntas de respuestas cerradas. Los resultados fueron presentados según su frecuencia absoluta y relativa. Resultados: Se entrevistaron a 77 jefes de familia, de los cuales 63,64 % (n=49) refirió tener antecedente de malaria y 38,96 % (n=30) conoció a alguien que haya fallecido en su comunidad por esta. En cuanto a conocimientos, 79,22 % (n=61) sabe que la enfermedad se adquiere por la picadura de un mosquito, 51,95 % (n=40) cree que se transmite por el agua y 97,4 % (n=75) asocia la fiebre como síntoma principal. A nivel de actitudes y prácticas, el 92,21 % (n=71) acudiría al médico para tratar la enfermedad, 62,34 % (n=29) mantiene los canales y zanjas limpias y 70,13 % (n=54) usa mosquitero y/o repelente. Conclusión: El área de Maniapure se ubica en una región endémica, y al tratarse de poblaciones susceptibles con diferente trasfondo cultural, se deben elaborar planes educativos individualizados orientados a la prevención, y evaluar su impacto en la práctica.

Introduction: Malaria is a disease with public health significance, with an increase in the last years in Bolivar State. As a potentially life-threatening infection, the development of preventive strategies based on community knowledge and practices is necessary. Objectives: To identify the knowledge, attitudes, and practices related to malaria and compare it with findings from other endemic areas. Methods: An observational, descriptive, and cross-sectional study was conducted in the Maniapure region from August 28 to 31, 2018. A questionnaire of 30 questions with closed answers was applied. The results were presented according to their absolute and relative frequency. Results: 77 family leaders were interviewed, of which 63.64 % (n=49) reported having a history of malaria and 38.96 % (n=30) knew someone who had died in their community from it. About knowledge, 79.22 % (n=61) knew that the disease is acquired by a mosquito bite, 51.95 % (n=40) believed that it's transmitted through water, and 97.4 % (n=75) associate fever as the main symptom. In relation to attitudes and practices, 92.21 % (n=71) would go to the doctor to treat the disease, 62.34 % (n=29) kept the channels and ditches clean and 70.13 % (n= 54) used a mosquito net and/or repellent. Conclusion: Maniapure is located in an endemic region, and since they have susceptible population with different cultural backgrounds, individualized educational plans (focused on prevention) should be elaborated, as well as test the impact in practices.

Evaluation of the transcriptional immune biomarkers in peripheral blood from Warao indigenous associate with the infection by Mycobacterium tuberculosis.

INTRODUCTION: Biomarkers are critical tools for finding new approaches for controlling the spread of tuberculosis (TB), including for predicting the development of TB therapeutics, vaccines, and diagnostic tools. METHODS: Expression of immune biomarkers was analyzed in peripheral blood cells stimulated and non-stimulated with M. tuberculosis antigens ESAT-6, CFP10 and TB7.7. in Warao indigenous individuals. These biomarkers may be able to differentiate TB states, such as active tuberculosis (ATB) cases and latent tuberculosis infection (LTBI) from non-infected controls (NIC). A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay was performed on 100 blood samples under non-stimulation or direct ex vivo conditions (NS=50) and stimulation conditions (S=50). RESULTS: The findings are shown as the median and interquartile range (IQR) of relative gene expression levels of IFN-γ, CD14, MMP9, CCR5, CCL11, CXCL9/MIG, and uPAR/PLAUR immune biomarkers. MMP9 levels were significantly higher in the LTBI-NS and LTBI-S groups compared with the NIC-NS and NIC-S groups. However, CCR5 levels were significantly lower in the LTBI-S group compared with both NIC-NS and NIC-S groups. CCL11 levels were significantly lower in the LTBI-S group compared with the NIC-NS group. CONCLUSIONS: Preliminary findings showed that MMP9 immune biomarkers separated LTBI indigenous individuals from NIC indigenous individuals, while CCR5, CCL11, CD14, and IFN-γ did not differentiate TB states from NIC. MMP9 may be useful as a potential biomarker for LTBI and new infected case detection among Warao indigenous individuals at high risk of developing the disease. It may also be used to halt the epidemic, which will require further validation in larger studies.

Evaluation of the transcriptional immune biomarkers in peripheral blood from warao indigenous associate with the infection by mycobacterium tuberculosis

Abstract INTRODUCTION: Biomarkers are critical tools for finding new approaches for controlling the spread of tuberculosis (TB), including for predicting the development of TB therapeutics, vaccines, and diagnostic tools. METHODS: Expression of immune biomarkers was analyzed in peripheral blood cells stimulated and non-stimulated with M. tuberculosis antigens ESAT-6, CFP10 and TB7.7. in Warao indigenous individuals. These biomarkers may be able to differentiate TB states, such as active tuberculosis (ATB) cases and latent tuberculosis infection (LTBI) from non-infected controls (NIC). A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay was performed on 100 blood samples under non-stimulation or direct ex vivo conditions (NS=50) and stimulation conditions (S=50). RESULTS: The findings are shown as the median and interquartile range (IQR) of relative gene expression levels of IFN-γ, CD14, MMP9, CCR5, CCL11, CXCL9/MIG, and uPAR/PLAUR immune biomarkers. MMP9 levels were significantly higher in the LTBI-NS and LTBI-S groups compared with the NIC-NS and NIC-S groups. However, CCR5 levels were significantly lower in the LTBI-S group compared with both NIC-NS and NIC-S groups. CCL11 levels were significantly lower in the LTBI-S group compared with the NIC-NS group. CONCLUSIONS: Preliminary findings showed that MMP9 immune biomarkers separated LTBI indigenous individuals from NIC indigenous individuals, while CCR5, CCL11, CD14, and IFN-γ did not differentiate TB states from NIC. MMP9 may be useful as a potential biomarker for LTBI and new infected case detection among Warao indigenous individuals at high risk of developing the disease. It may also be used to halt the epidemic, which will require further validation in larger studies.

Concordance between IFNγ gene +874 A/T polymorphism and interferon-γ expression in a TB-endemic indigenous setting.

INTRODUCTION:: Interferon-γ (IFN-γ) plays a crucial role in resistance to mycobacterial diseases; accordingly, variants of the gene encoding this cytokine may be associated with elevated risk of contracting pulmonary tuberculosis (TB). METHODS:: Blood samples were collected from 135 Warao indigenous individuals with newly diagnosed sputum culture-positive TB. Of these, 24 were diagnosed with active tuberculosis (ATB). The study comprised 111 participants, who were grouped as follows: 1) 14 tuberculin skin test (TST)-positive Warao indigenous individuals and 4 that were QuantiFERON-TB?Gold In-Tube (QFT-IT) test-positive, collectively comprising the latent TB infection group (LTBI), n = 18), and 2) healthy controls who were QFT-IT- and TST-negative, comprising the control group (CTRL, n = 93). Detection of the IFN γ gene (IFNG) +874A/T polymorphism was performed via PCR and quantification of IFNG expression via qPCR. RESULTS:: Relative to indigenous and white Americans, ATB and CTRL groups had a higher frequency of the IFNG SNP (+874A): 23 (95.8%) and 108 (97.3%), respectively. Indigenous Warao individuals homozygous for the IFNG (+874) A allele exhibited 3.59-fold increased risk of developing TB (95% confidence interval, 2.60-4.96, p =0.0001). A decreased frequency of the AT genotype was observed in individuals with pulmonary TB (4.16%) and controls (0.90%). The frequency of the TT genotype was decreased among controls (1.80%); none of the patients with TB were found to have this genotype. The differences in IFNG expression between the groups, under unstimulated and stimulated conditions, were not statistically significant. CONCLUSIONS:: Preliminary results demonstrate concordance between IFNG +874 A/A genotype and low expression of IFNG.

Concordance between IFNγ gene +874 A/T polymorphism and interferon-γ expression in a TB-endemic indigenous setting

Abstract INTRODUCTION: Interferon-γ (IFN-γ) plays a crucial role in resistance to mycobacterial diseases; accordingly, variants of the gene encoding this cytokine may be associated with elevated risk of contracting pulmonary tuberculosis (TB). METHODS: Blood samples were collected from 135 Warao indigenous individuals with newly diagnosed sputum culture-positive TB. Of these, 24 were diagnosed with active tuberculosis (ATB). The study comprised 111 participants, who were grouped as follows: 1) 14 tuberculin skin test (TST)-positive Warao indigenous individuals and 4 that were QuantiFERON-TB?Gold In-Tube (QFT-IT) test-positive, collectively comprising the latent TB infection group (LTBI), n = 18), and 2) healthy controls who were QFT-IT- and TST-negative, comprising the control group (CTRL, n = 93). Detection of the IFN γ gene (IFNG) +874A/T polymorphism was performed via PCR and quantification of IFNG expression via qPCR. RESULTS: Relative to indigenous and white Americans, ATB and CTRL groups had a higher frequency of the IFNG SNP (+874A): 23 (95.8%) and 108 (97.3%), respectively. Indigenous Warao individuals homozygous for the IFNG (+874) A allele exhibited 3.59-fold increased risk of developing TB (95% confidence interval, 2.60-4.96, p =0.0001). A decreased frequency of the AT genotype was observed in individuals with pulmonary TB (4.16%) and controls (0.90%). The frequency of the TT genotype was decreased among controls (1.80%); none of the patients with TB were found to have this genotype. The differences in IFNG expression between the groups, under unstimulated and stimulated conditions, were not statistically significant. CONCLUSIONS: Preliminary results demonstrate concordance between IFNG +874 A/A genotype and low expression of IFNG.

Prevalence of asymptomatic Plasmodium vivax infections in the north-eastern focus of malaria of Venezuela / Prevalencia de infecciones asintomáticas por Plasmodium vivax en el foco malárico oriental de Venezuela

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.

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.

Seropositivity for ascariosis and toxocariosis and cytokine expression among the indigenous people in the Venezuelan Delta region.

The present study aimed at measuring seropositivities for infection by Ascaris suum and Toxocara canis using the excretory/secretory (E/S) antigens from Ascaris suum (AES) and Toxocara canis (TES) within an indigenous population. In addition, quantification of cytokine expressions in peripheral blood cells was determined. A total of 50 Warao indigenous were included; of which 43 were adults and seven children. In adults, 44.1% were seropositive for both parasites; whereas children had only seropositivity to one or the other helminth. For ascariosis, the percentage of AES seropositivity in adults and children was high; 23.3% and 57.1%, respectively. While that for toxocariosis, the percentage of TES seropositivity in adults and children was low; 9.3% and 14.3%, respectively. The percentage of seronegativity was comparable for AES and TES antigens in adults (27.9%) and children (28.6%). When positive sera were analyzed by Western blotting technique using AES antigens; three bands of 97.2, 193.6 and 200.2 kDas were mostly recognized. When the TES antigens were used, nine major bands were mostly identified; 47.4, 52.2, 84.9, 98.2, 119.1, 131.3, 175.6, 184.4 and 193.6 kDas. Stool examinations showed that Blastocystis hominis, Hymenolepis nana and Entamoeba coli were the most commonly observed intestinal parasites. Quantification of cytokines IFN-γ, IL-2, IL-6, TGF-ß, TNF-α, IL-10 and IL-4 expressions showed that there was only a significant increased expression of IL-4 in indigenous with TES seropositivity (p < 0.002). Ascaris and Toxocara seropositivity was prevalent among Warao indigenous.

Seropositivity for ascariosis and toxocariosis nd cytokine expression among the indigenous people in the Venezuelan delta region / Seropositividad para ascariosis y toxocariosis y expresión de citocinas entre la población indígena de la región del delta Venezolano

The present study aimed at measuring seropositivities for infection by Ascaris suum and Toxocara canis using the excretory/secretory (E/S) antigens from Ascaris suum (AES) and Toxocara canis (TES) within an indigenous population. In addition, quantification of cytokine expressions in peripheral blood cells was determined. A total of 50 Warao indigenous were included; of which 43 were adults and seven children. In adults, 44.1% were seropositive for both parasites; whereas children had only seropositivity to one or the other helminth. For ascariosis, the percentage of AES seropositivity in adults and children was high; 23.3% and 57.1%, respectively. While that for toxocariosis, the percentage of TES seropositivity in adults and children was low; 9.3% and 14.3%, respectively. The percentage of seronegativity was comparable for AES and TES antigens in adults (27.9%) and children (28.6%). When positive sera were analyzed by Western blotting technique using AES antigens; three bands of 97.2, 193.6 and 200.2 kDas were mostly recognized. When the TES antigens were used, nine major bands were mostly identified; 47.4, 52.2, 84.9, 98.2, 119.1, 131.3, 175.6, 184.4 and 193.6 kDas. Stool examinations showed that Blastocystis hominis, Hymenolepis nana and Entamoeba coli were the most commonly observed intestinal parasites. Quantification of cytokines IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 and IL-4 expressions showed that there was only a significant increased expression of IL-4 in indigenous with TES seropositivity (p < 0.002). Ascaris and Toxocara seropositivity was prevalent among Warao indigenous.

El objetivo del presente estudio fue determinar la seropositividad de infección por Ascaris suum y Toxocara canis, utilizando antígenos de excreción/secreción (E/S) de Ascaris suum (AES) y Toxocara canis (TES) en una población indígena. Adicionalmente, se cuantificó la expresión de citocinas a partir de células de sangre periférica. Un total de 50 indígenas Warao se incluyeron en el estudio; 43 fueron adultos y 7 niños. Entre los adultos, 44,1% fueron seropositivos para ambos parásitos; mientras que los niños sólo mostraron seropositividad a uno u otro de los helmintos. Para ascariosis, el porcentaje de seropositividad para los antígenos AES fue alto tanto en adultos como en niños; 23,3% y 57,1%, respectivamente. Para toxocariosis, el porcentaje de seropositividad para los antígenos TES fue bajo en adultos así como en niños; 9,3% y 14,3%, respectivamente. El porcentaje de seronegatividad fue similar tanto para los antígenos AES como para TES en adultos (27,9%) y niños (28,6%). Cuando la seropositividad fue analizada a través de la técnica de Western blotting utilizando los antígenos AES; 3 bandas de 97,2, 193,6 y 200,2 kDas fueron principalmente reconocidas. Para los antígenos TES, 9 bandas fueron mayormente identificadas; 47,4, 52,2, 84,9, 98,2, 119,1, 131,3, 175,6, 184,4 y 193,6 kDas. Los análisis coproparasitológicos mostraron que los parásitos Blastocystis hominis, Hymenolepis nana y Entamoeba coli fueron los parásitos intestinales más comúnmente observados. La cuantificación de la expresión de las citocinas IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 e IL-4 mostró que hubo un significante incremento de la expresión de IL-4 entre los indígenas con seropositividad para los antígenos TES (p < 0.002). La seropositividad para Ascaris y Toxocara fue prevalente entre los indígenas Warao.

Patterns of selection on Plasmodium falciparum erythrocyte-binding antigens after the colonization of the New World.

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte-binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba-140, eba-175, eba-181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba-140 seemed to be under stronger diversifying selection in South America than eba-175. In contrast, eba-181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins.

Patients exposed to Mycobacterium tuberculosis infection with a prominent IgE response.

BACKGROUND AND AIMS: Even though it has been reported that chronic immune activation associated with intestinal helminthic infections results in a predominant IgE response, specific IgE antibodies that are also interleukin 4 (IL-4) dependent have been reported in tuberculosis patients; however, this fact has not been widely reported. This study was aimed at investigating the levels of circulating IgE in Warao (an indigenous population) of the Orinoco river delta, an area isolated from contact with the tubercle bacillus for millennia until the mid-1960s as compared to Creole (nonindigenous population). METHODS: A total of 294 individuals were studied, 161 Warao and 136 Creole. Patient group was comprised of 86 Warao patients (WP) and 60 Creole patients (CP). Control group was comprised of 75 Warao controls (WC) and 76 Creole controls (CC). Total serum IgE and IgE and IgG(4) reactivities to M. tuberculosis antigens were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Levels of total serum IgE were significantly elevated in WP (13002.0 ± 11200.0 IU/mL) and WC (2763.5 ± 2596.2 IU/mL) than in CP (385.9 ± 155.1 IU/mL) and CC (356.6 ± 157.5 IU/mL) (p <0.0001). Anti-PPD and anti-H37Rv IgE were significantly higher in WP (0.240 ± 0.145 and 0.230 ± 0.155) than in CP (0.127 ± 0.152 and 0.97 ± 0.103, respectively) and also between WC (0.240 ± 0.273 and 0.147 ± 0.158) and CC (0.115 ± 0.136 and 0.43 ± 0.46, respectively) (p <0.0001). Anti-PPD and anti-H37Rv IgG(4) did not show differences among groups; however, anti-H37Rv IgG(4) was affected by anti-TB treatment, which could be predictive of treatment outcome. CONCLUSIONS: The findings suggest that for the Warao population there is an intrinsic propensity to produce a high IgE response, which could be incompatible with the protective response to M. tuberculosis.

Multiple independent introductions of Plasmodium falciparum in South America.

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Antígenos de Plasmodium falciparum, blancos de la respuesta inmunitaria humoral en pacientes tratados con cloroquina / Antigens of Plasmodium falciparum, target of humoral immune response in patients treated with chloroquine

Se planteó identificar antígenos que pudieran ser reconocidos por los anticuerpos IgG1 e IgG3, descritos como protectores en la infección malárica, en personas con respuesta clínica adecuada (RCA) o falla al tratamiento (FT) antimalárico, provenientes de localidades con diferentes grados de endemicidad. Se evaluaron por Immunoblotting muestras de sueros de individuos provenientes de tres localidades del Edo. Amazonas (Venezuela): Puerto Ayacucho (Atures), San Juan de Manapiare (Manapiare) y Platanal (Alto Orinoco). La reactividad de IgG, IgG1 e IgG3 frente a componentes antigénicos del extracto de P. falciparum (FCB2), permitió identificar un mayor número de moléculas específicas en los pacientes con RCA que en los pacientes con FT. La frecuencia de reconocimiento de polipéptidos fue baja en las tres localidades, algunas moléculas con una frecuencia de reconocimiento igual o mayor al 20% pertenecían a sueros de individuos de las localidades de Puerto Ayacucho y Platanal, ambas con exposición permanente a P. falciparum. Dado el reconocimiento de polipéptidos por IgG, IgG1 e IgG3 en sueros de pacientes con RCA, estos podrían ser considerados como posibles blancos relevantes de la respuesta inmunológica protectora que coadyuven con el tratamiento antimalárico. Esto contribuiría al desarrollo y diseño de vacunas más efectivas, que prevengan la infección malárica y/o potencien la eficacia a la quimioterapia.

Here we studied the presence of antigens recognized by IgG1 and IgG3 antibodies, thought as protective, in patients with adequate clinical response (RCA) or treatment failure (FT), living in areas of different degrees of endemicity. Immunoblotting was evaluated from serum samples of individuals from three locations in the State Amazonas (Venezuela): Puerto Ayacucho (Atures), San Juan de Manapiare (Manapiare) and Pantanal (Alto Orinoco). The reactivity of IgG, IgG1 and IgG3 against antigenic components of the extract of P. falciparum (FCB2) identified a greater number of specific molecules in patients with RCA in patients with AFT. The frequency of recognition of polypeptides was low in all three locations, with some molecules having a recognition rate of greater than or equal to 20% sera of individuals belonging to the towns of Puerto Ayacucho and Platanal, both with cases of P. falciparum. Given the recognition of polypeptides by IgG, IgG1 and IgG3 in sera of patients with RCA, they could be considered as possible targets for relevant protective immune responses that contribute to malaria treatment. This would contribute to the development and design of more effective vaccines that prevent malaria infection and/or enhance the efficacy of chemotherapy.

Comparison of serological responses in two different populations with pulmonary tuberculosis.

Observational studies on the humoural immune responses of the Warao indigenous people from Delta Amacuro, an isolated area, were compared with urban residents of the Venezuelan capital. Mycobacterium tuberculosis-specific reactivities (IgM, IgE, sIgA, IgG and IgG subclasses) were measured by ELISA using PPD and 38-kDa M. tuberculosis antigens. A total of 294 individuals were studied, 162 Warao (indigenous people) and 132 Creole (non-indigenous people). The patient group consisted of 87 Warao patients and 58 Creole patients, while the control group consisted of 75 Warao controls and 74 Creole controls. Combinations among the isotypes studied were performed. The findings showed that for the Warao people, sensitivity to the combination including anti-PPD IgG and IgE was 92.0%, while for the Creole people, sensitivity to the combination including anti-PPD IgG but more so anti-PPD IgG1 and IgG2 was 90.0%. Simple tests were able to show higher specificities, which were population-specific; specificities were anti-PPD IgG3, 100.0% and anti-PPD IgM, 97.4% for the Warao and Creole peoples, respectively. In conclusion, while simple tests reached high specificity, the multi-isotype tests improved sensitivity; the latter shows this approach may be useful in diagnostic testing.

Comparison of serological responses in two different populations with pulmonary tuberculosis

Observational studies on the humoural immune responses of the Warao indigenous people from Delta Amacuro, an isolated area, were compared with urban residents of the Venezuelan capital. Mycobacterium tuberculosis-specific reactivities (IgM, IgE, sIgA, IgG and IgG subclasses) were measured by ELISA using PPD and 38-kDa M. tuberculosis antigens. A total of 294 individuals were studied, 162 Warao (indigenous people) and 132 Creole (non-indigenous people). The patient group consisted of 87 Warao patients and 58 Creole patients, while the control group consisted of 75 Warao controls and 74 Creole controls. Combinations among the isotypes studied were performed. The findings showed that for the Warao people, sensitivity to the combination including anti-PPD IgG and IgE was 92.0 percent, while for the Creole people, sensitivity to the combination including anti-PPD IgG but more so anti-PPD IgG1 and IgG2 was 90.0 percent. Simple tests were able to show higher specificities, which were population-specific; specificities were anti-PPD IgG3, 100.0 percent and anti-PPD IgM, 97.4 percent for the Warao and Creole peoples, respectively. In conclusion, while simple tests reached high specificity, the multi-isotype tests improved sensitivity; the latter shows this approach may be useful in diagnostic testing.

Evaluation of the OptiMAL test for diagnosis of malaria in Venezuela.

We evaluated the OptiMAL rapid dipstick test by comparing it with the conventional standard thick-blood film method, for the detection of malaria in two groups of individuals from different Venezuelan endemic areas. One of the groups consisted of individuals with malaria-like symptoms (n = 113) and the other of asymptomatic individuals (n = 89). The classical microscopy analysis of these populations determined that 67.5% were infected with P. vivax, 31.3% with P. falciparum, and 1.2% with mixed infections. The OptiMAL test showed 96.4% sensitivity, 100% specificity, 100% positive predictive value, 97.5% negative predictive value and optimal concordance (kappa = 0.97), capable of detecting any malaria infection in the evaluated population. However, these parameters were lower when the parasitaemia was < or = 300 parasites/microL. Freezing of the samples did not affect the sensitivity and specificity of the test. We concluded that this rapid malaria test is sensitive and specific for rapid diagnosis of malaria in the field and it is a complement to conventional microscopy.

Evaluation of the OptiMAL® test for diagnosis of malaria in Venezuela

Se evaluó el test rápido OptiMAL® comparándolo con el examen convencional de observación de muestras sanguíneas para la detección de la malaria en dos grupos de individuos procedentes de diferentes áreas endémicas de Venezuela. Uno de los grupos (n = 113) con síntomas sugestivos de malaria, y otro representado por individuos asintomáticos (n = 89). El examen microscópico de las muestras de sangre de estas poblaciones determinó que 67,5 por ciento estaban infectados con P.vivax, 31,3 por ciento con P. falciparum y 1,2 por ciento con infecciones mixtas. La prueba OptiMAL® mostró 96,4 por ciento de sensibilidad, 100 por ciento de especificidad, con valor predictivo positivo de 100 por ciento y valor predictivo negativo de 97,5 por ciento. La detección de cualquier infección malárica en la población total presentó una concordancia óptima (kappa = 0,97). Sin embargo, estos parámetros fueron más bajos cuando la parasitemia era £ 300 parásitos/µL. El congelamiento de las muestras no afectó la sensibilidad y especificidad de la prueba. Nosotros concluimos que esta prueba rápida de malaria es sensible y especifica para el diagnóstico rápido de la malaria en el campo y puede complementar a la microcopia convencional.

Evaluación de la prueba NOW® ICT Pf/Pv para el diagnóstico de malaria en Venezuela / Evaluation of the NOW® ICT P.f/P.v. test for the diagnosis of malaria in Venezuela

Se evaluó la prueba NOW® malaria ICT P.f/P.v. en personas con síntomas de malaria provenientes de dos áreas andémicas de Venezuela: estado Sucre en la región noreste costera (n=71) y el estado Amazonas en el sur (n=86). 102 muestras resultaron positivas (73 P. vivax, 25 P. falciparum, 2 P. malariae y 2 infecciones mixtas), 55 muestras resultaron negativas mediante el exámen microscópico. La sensibilidad, especificidad, VPP y VPN del NOW® malaria ICT P.f/P.v. para el diagnóstico de la malaria fue 81,4%, 100%, 100% y 74,3%, respectivamente y un Kappa de 0,75. En Sucre la sensibilidad fue de 96,3%, especificidad 100%, VPP 100%, VPN 90,5% y Kappa 0,85 para P. vivax. En Amazonas, la sensibilidad fue 81%, especificidad y VPP 100%, VPN 90,5% y Kappa 0,85 para infecciones activas por P.vivax y P. malariae. Esta sensibilidad para P. vivax aumenta con parasistemias entre 101-500 par/µL. La concordancia encontrada en este estudio entre la prueba Now®malaria ICT P.f/P.v. y el método parasitológico para las especies P. vivax/P. malariae, aunque no para P. falcifarum, aporta un valor adicional a las ya conocidas características de la prueba tales como procedimiento fácil, rapidez e interpretación de los resultados en áreas de díficil acceso, por lo que concluimos que es una herramienta alternativa para el diagnóstico de malaria, sin llegar a sustituir el método parasitológico convencional.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Epítopes de antígenos compartidos entre Plasmodium falciparum y Anopheles albimanus fueron identificados. Diferentes grupos de conejos fueron inmunizados con: extracto crudo de mosquito hembra de An. albimanus (EAaH), glóbulos rojos infectados con P. falciparum (EPfs) y la vacuna antimalárica sintética SPf66. Los anticuerpos policlonales producidos en conejos fueron evaluados por ELISA, inmunoensayo simultáneo de múltiples antígenos (MABA) e Immunoblotting. Todos los extractos resultaron inmunogénicos cuando se evaluaron por ELISA, MABA e Immunoblotting. Diez moléculas fueron identificadas en los mosquitos hembras y diez en los antígenos de P. falciparum por los sueros autólogos. El patrón electroforético por SDS-EGPA fue diferente para los tres antígenos evaluados. La reactividad cruzada de moléculas entre An. albimanus y P. falciparum fue demostrada por ELISA, MABA e Immunoblotting. Anticuerpos anti-P. falciparum y anti-SPf66 reconocieron diez y cinco componentes respectivamente en el extracto crudo de anofelinos (EAaH). Asimismo, sueros inmunes contra An. albimanus hembra identificaron cuatro moléculas en el extracto del antígeno de P. falciparum. Hasta el presente, este es el primer estudio en el que se demuestra la presencia de antígenos compartidos entre anofelinos y los parásitos de malaria. Este hallazgo podría ser de relevancia para el diagnóstico, vacunas e interpretación de la fisiopatología de la respuesta inmunitaria en malaria.

Immunogenicity of synthetic peptides derived from Plasmodium falciparum proteins.

To obtain antibodies suitable to be used in an antigen-capture assay, we have identified, synthesized, and evaluated a series of peptides from different Plasmodium falciparum excretory-secretory proteins: glutamate-rich protein (GLURP); histidine-rich protein 2; histidine-rich protein 3; Falciparum interspersed repeat antigen and, serine-rich antigen homologous. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in rabbits. Out of the 14 peptide constructs, eight by ELISA and, six by MABA elicited antibodies that showed correspondence between the predictive study and the immunogenicity obtained in rabbits. All antipeptide (GLURP, HRP2, and FIRA) antisera were found to bind to the corresponding synthetic sequence in an ELISA assay. The binding activity and specificity of antibodies were determined by Western blot with supernatant culture from P. falciparum. Anti-GLURP (IMT-94 and IMT-200) antisera bound to five molecules present in supernatant with molecular weight of 73, 82, 116, 124, and 128 kDa. Anti-HRP2 (IMT-192) antisera recognized a band of 58 kDa. In both cases, the specific molecules were inhibited by preincubation with the homologous peptide. Anti-HRP3, anti-FIRA neither anti-SERPH antisera showed reactivity. Anti-peptides HRP2 antibodies recognized the recombinant protein present in Parasight-F test. The same way, synthetic peptides from HRPII molecule were recognized by monoclonal antibody present in the Parasight-F assay. Our results confirm the potential value of synthetic peptides when inducing monospecific polyclonal antibodies for the development of diagnostic tests based on the capture of antigens.