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.

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.

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.

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.

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.

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.

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% estaban infectados con P.vivax, 31,3% con P. falciparum y 1,2% con infecciones mixtas. La prueba OptiMAL® mostró 96,4% de sensibilidad, 100% de especificidad, con valor predictivo positivo de 100% y valor predictivo negativo de 97,5%. 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.

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 £ 300 parasites/µL. 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.

Se evaluó la prueba Now® malaria ICT P.f/P.v. en personas con síntomas de malaria provenientes de dos áreas endémicas 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 examen microscópico. La sensibilidad, especificidad, VPP y VPN del Now® malaria ICT P.f/P.v. para el diagnóstico de 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 89,5% y Kappa 0,93 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 parasitemias entre 101-500 par/μL. Para P. falciparum la sensibilidad fue 51,9%, especificidad de 100%, VPP de 100%, VPN de 74,5% y un Kappa de 0,56. La sensibilidad del método es de 100% para densidades mayores a 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. falciparum, 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 difícil acceso, por lo que concluimos que es una herramienta alternativa para el diagnóstico de malaria, sin llegar a sustituir el método parasitológico convencional.

We evaluated the Now® malaria ICT P.f/ P.v. test in symptomatic malaria patients from two different endemic areas of Venezuela: Sucre state in the northeastern costal region (n=71) and the Amazonas state in the south (n=86). One hundred two patients were positive by thick and thin blood smears, 73 with Plasmodium vivax , 25 with P. falciparum , 2 with P. malariae and 2 with mixed infections. Some 55 blood samples were negative. The sensitivity, specificity, PPV, NPV of the Now® malaria ICT P.f/P.v. test, were 81.4%, 100%, 100% and 74.3%, respectively and a Kappa of 0.75. With blood samples from Sucre state, the sensitivity was 96.3%, the specificity 100%, the PPV 100%, NPV 89.5% and a Kappa of 0.93 for P. vivax. In Amazonas State, the sensitivity was 81%, the specificity and PPV 100%, the NPV 90.5% and a Kappa of 0.85 for P. vivax and P. malariae. This sensitivity increased for P. vivax when parasitaemias were above 101 parasites/uL. For P. falciparum , the sensitivity was 51.9%, the specificity 100%, the PPV 100%, NPV was 74.5% and a Kappa of 0.56. The sensitivity was 100%, with more than 500 parasites/mL for this species. The high concordance found in this test with the thick and thin blood smear for all malaria species except P. falciparum gives an additional value to this dipstick, along with other well known characteristics such as the rapid and easy handling and interpretation in areas of difficult access. We concluded that the immunochromatographic test is a valuable additional diagnostic tool for malaria but not a substitute of the classical thin and thick blood smear.

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.