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.

Diagnostic Potential of the Serological Response to Synthetic Peptides from Mycobacterium tuberculosis Antigens for Discrimination Between Active and Latent Tuberculosis Infections.

Diagnosis and treatment of active tuberculosis (ATB) as well as latent tuberculosis infection (LTBI) are required for effective tuberculosis (TB) control, especially in TB endemic area. The usefulness of conventional tests to distinguish between ATB and LTBI has remained challenging. The present study was aimed to demonstrate the usefulness of the serological response to synthetic peptides from Mycobacterium tuberculosis (Mtb) antigens for discrimination between ATB and LTBI in Warao Amerindians. Serum IgG antibody levels were measured by the indirect ELISA assay using 22 designed and synthesized peptides derived from immunogenic Mtb ESAT-6 and Ag85A proteins. A total of 211 adult Warao Amerindians were included; cases with active TB (ATB, n = 75), latent TB infection (LTBI, n = 85) and non-infected (NI, n = 51). The approach's diagnostic information was compared using receiver operating characteristic (ROC) curves. For ATB diagnostic performance between ATB and NI; ESAT-6; P-12037 had 100% of sensitivity (AUC = 0.812; 0.733 to 0.891 95% CI); and Ag85A; P-10997 had 100% of specificity (AUC = 0.691; 0.597 to 0.785 95% CI); and ATB and LTBI; Ag85A; P-29878 had 100% of sensitivity (AUC = 0.741; 0.666-0.817 95% CI), and P-29879 had 99% of specificity (AUC = 0.679; 0.593-0.765 95% CI). While that ESAT-6 P-12037 also allowed differentiation between LTBI and NI or healthy ones. It had 98.8% of sensitivity and 98.0% of specificity (AUC = 0.640; 0.545-0.735 95% CI). The potential of combination-antigen immunoassays with peptides could discriminate between Warao Amerindians with ATB, LTBI and NI. Further validation of this approach could lead to developing a complementary tool for rapid diagnosis of TB infections.

Nicotine associates to intracellular Mycobacterium tuberculosis inducing genes related with resistance to antimicrobial peptides.

Tobacco consumption is related to an increased risk to develop tuberculosis. Antimicrobial peptides are essential molecules in the response to Mycobacterium tuberculosis (Mtb) because of their direct antimicrobial activity. The aim of this study was to demonstrate that nicotine enters into Mtb infected epithelial cells and associates with the mycobacteria inducing genes related to antimicrobial peptides resistance. Epithelial cells were infected with virulent Mtb, afterwards cells were stimulated with nicotine. The internalization of nicotine was followed using electron and confocal microscopy. The lysX expression was evaluated isolating mycobacterial RNA and submitted to RT-PCR analysis. Our results indicated that nicotine promotes Mtb growth in a dose-dependent manner in infected cells. We also reported that nicotine induces lysX expression. In conclusion, nicotine associates to intracellular mycobacteria promoting intracellular survival.

ESAT-6 and Ag85A Synthetic Peptides as Candidates for an Immunodiagnostic Test in Children with a Clinical Suspicion of Tuberculosis.

BACKGROUND: Tuberculosis (TB) is being underdetected in children as most are smear-negative. This work was aimed at evaluating ESAT-6 and Ag85A synthetic peptides' serodiagnostic potential for diagnosing children having a clinical suspicion of TB. METHODS: The study involved 438 children: 77 Creole nonindigenous (13 suspected of having TB and 64 healthy ones) and 361 Warao indigenous children (39 suspected of TB and 322 healthy children). The approach's diagnostic information was compared using operational characteristics and receiver-operating characteristic (ROC) curves. RESULTS: Ag85A P-29879 had 94.6% sensitivity (AUC = 0.741: 0.651 to 0.819 95% CI) in indigenous children. ESAT-6 P-12036 and P-12037 had 100% and 92.3% of sensitivity (AUC = 0.929: 0.929: 0.846 to 0.975 95% CI and 0.791: 63.9 to 98.7 95% CI, respectively) in Creole children. ESAT-6 peptides also allowed a differentiation between children with TB and healthy ones. CONCLUSIONS: Further validation of this approach could lead to developing a complementary tool for rapid TB diagnosis in children.

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.

Diagnostic accuracy of combinations of serological biomarkers for identifying clinical tuberculosis.

INTRODUCTION: Confirmation of tuberculosis (TB) cases in endemic TB settings is a challenge; obtaining fast and cheap, though accurate, diagnostic tools such as biomarkers is thus urgently needed to enable the early detection of TB. This paper evaluates the diagnostic accuracy of combinations of host serological biomarkers for identifying TB. METHODOLOGY: Enzyme-linked immunosorbent assays (ELISA) were used on 70 Venezuelan Creole individuals for evaluating host biomarkers (i.e. CXCL9, sCD14, MMP9 and uPAR proteins) and anti-synthetic peptides covering certain Mycobacterium tuberculosis (Mtb) ESAT-6 (P-12033, P-12034 and P-12037) and Ag85A (P-29878) antigen sequences. The target population consisted of adults having active TB (ATB, n = 28), the tuberculin skin test positive (TST+) or individuals with latent TB infection (LTB, n = 28) and TST- or control subjects (CTRL, n = 14). RESULTS: Receiver operator curve (ROC) analysis revealed good biosignature discriminative ability for 5 serological biomarkers; the accuracy of 3 combinations had a good discriminative ability for diagnosing TB. Anti-P-12034/uPAR detected TB with 96.7% sensitivity and 86.0% specificity, followed by anti-P-12033/uPAR having 96.7% sensitivity and 81.4% specificity. Anti-P-29878/MMP9 had the highest sensitivity (100%), but low specificity (52.17%). Biomarker combinations did not prove efficacious for identifying incipient subclinical TST+TB- subjects at high-risk for TB. CONCLUSIONS: The anti-P-12034/uPAR combination could be useful for identifying clinical TB patients. Such an approach holds promise for further validation.

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.

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.

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection.

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.

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.

Diagnostic accuracy of immunological methods in patients with tuberculous pleural effusion from Venezuela.

In recent years, better diagnostics for tuberculosis (TB) has received increasing attention, especially the diagnosis of tuberculous pleural effusion, which is difficult and at present the main tool in TPE diagnostic is pleural effusion smear and culture, but unfortunately, sensitivities are low, therefore better TPE diagnostic tools are needed. The aim of this study was to find a diagnostic algorithm to assess the progress in TPE diagnostic at the Hospital Vargas de Caracas, that permits identification of the majority of patients, at a satisfactory cost-benefit ratio, evaluating the levels of IFN-gamma and IL-12p40 in pleural effusion and serum, as well as the antibody reactivity in order to compare it with microbiological tests. A total of 60 individuals with pleural effusion were studied; 20 patients with tuberculous pleural effusion (TPE) formed the patient group and 40 patients with non-tuberculous pleural effusion (NTPE) formed the control group. The levels of IFN-gamma and IL-12p40 in effusion and serum and class and subclasses of IgG reactivity to Mycobacterium tuberculosis antigens were measured by ELISA. The utility of these methods for diagnosis of TPE was evaluated using receiver operating characteristic (ROC) curve analysis. The results of the 11 immunological methods evaluated showed that the anti-PPD IgG2 method was able to reach the highest specificity of 95% (CI: 88.3-101.8), positive predictive value (PPV) = 75 (at 30% sensitivity); while that the overall sensitivity of methods was between 95% and 30%, of these, two methods reached higher sensitivities; increased levels of pleural IFN-gamma, with a sensitivity of 95% (CI: 85.5-104.5) with the highest negative predictive value (NPV) = 97, (at 82.5% specificity), followed by decreased levels of serum IL-12p40 with a sensitivity of 95% (CI: 85.5-104.5), NPV = 95.2 (at 50% specificity). In contrast, microbiological methods showed that smear had a sensitivity of only 20%, while smear plus culture had, a sensitivity of 70%. Considering that TPE represents approximately 15 percent of all the TB clinically diagnosed at the Hospital Vargas de Caracas, in those patients with preliminary microbiology negativity in the effusion, the combined analysis of pleural IFN-gamma and anti-PPD IgG2 could represent a fast and effective diagnostic algorithm for improving the diagnosis previous to obtain culture results. In this way treatment against TB could be initiated or the need to cytological and pleural biopsy could be considered.

Differential expression of antimicrobial peptides in active and latent tuberculosis and its relationship with diabetes mellitus.

Tuberculosis (TB) is one of the most important infectious diseases, causing 1.8 million deaths annually worldwide. This problem has increased because of the association with human immmunodeficiency virus and diabetes mellitus type 2, mainly in developing countries. In the past few years it has been highlighted the significance of antimicrobial peptides in the immunopathogenesis of TB ex vivo and in experimental models studies. In this study we analyzed the expression of CAMP, DEFA1, DEFB4, and DEFB103A in patients with latent TB and progressive TB with and without comorbidity with diabetes mellitus type 2. Antimicrobial peptide gene expression increased during progressive TB, which could be used as a biomarker for reactivation. By contrast, patients with diabetes mellitus type 2 have lower antimicrobial peptides gene expression, suggesting that the lack of its proper production in these patients contribute to enhance the risk for TB reactivation.

Diagnostic accuracy of immunological methods in patients with tuberculous pleural effusion from Venezuela / Precisión diagnóstica de métodos inmunológicos en pacientes con tuberculosis pleural de Venezuela

In recent years, better diagnostics for tuberculosis (TB) has received increasing attention, especially the diagnosis of tuberculous pleural effusion, which is difficult and at present the main tool in TPE diagnostic is pleural effusion smear and culture, but unfortunately, sensitivities are low, therefore better TPE diagnostic tools are needed. The aim of this study was to find a diagnostic algorithm to assess the progress in TPE diagnostic at the Hospital Vargas de Caracas, that permits identification of the majority of patients, at a satisfactory cost-benefit ratio, evaluating the levels of IFN-g and IL-12p40 in pleural effusion and serum, as well as the antibody reactivity in order to compare it with microbiological tests. A total of 60 individuals with pleural effusion were studied; 20 patients with tuberculous pleural effusion (TPE) formed the patient group and 40 patients with non-tuberculous pleural effusion (NTPE) formed the control group. The levels of IFN-g and IL-12p40 in effusion and serum and class and subclasses of IgG reactivity to Mycobacterium tuberculosis antigens were measured by ELISA. The utility of these methods for diagnosis of TPE was evaluated using receiver operating characteristic (ROC) curve analysis. The results of the 11 immunological methods evaluated showed that the anti-PPD IgG2 method was able to reach the highest specificity of 95% (CI: 88.3-101.8), positive predictive value (PPV)=75 (at 30% sensitivity); while that the overall sensitivity of methods was between 95% and 30%, of these, two methods reached higher sensitivities; increased levels of pleural IFN-g, with a sensitivity of 95% (CI: 85.5-104.5) with the highest negative predictive value (NPV)=97, (at 82.5% specificity), followed by decreased levels of serum IL-12p40 with a sensitivity of 95% (CI: 85.5-104.5), NPV=95.2 (at 50% specificity). In contrast, microbiological methods showed that smear had a sensitivity of only 20%, while smear plus ...

Recientemente existe un gran interés hacia un mejor y más rápido diagnóstico de tuberculosis (TB), especialmente de tuberculosis pleural, el cual es difícil. Al presente las principales herramientas diagnósticas son la baciloscopia y el cultivo de líquido pleural; desafortunadamente, las sensibilidades de estos métodos son bajas, por lo que el desarrollo de nuevas herramientas diagnósticas es necesario. El objetivo del presente estudio consistió en encontrar un algoritmo que permita la rápida identificación de la mayoría de los pacientes con TB pleural que ingresan en el Hospital Vargas de Caracas a un buen costo-beneficio. Para esto se evaluaron los niveles de las citocinas Interferón-gamma (IFN-g) y la Interleucina 12p40 (IL-12p40) en líquido pleural y suero, así como la reactividad de anticuerpos contra antígenos de Mycobacterium tuberculosis. Se estudiaron 60 individuos con derrame pleural; 20 individuos con líquido pleural tuberculoso (LPT) conformaron el grupo de pacientes y 40 individuos con líquido pleural no tuberculoso (LPNT) el grupo de controles. La técnica de inmunoensayo de ELISA fue utilizada para medir los niveles de IFN-g y IL-12p40; así como las reactividades de los diversos isotipos y subclases de inmunoglobulina G (IgG) frente a antígenos del bacilo. La utilidad de los métodos fue evaluada utilizando el análisis de las curvas ROC (receiver operating characteristic). Los resultados de los 11 métodos inmunológicos evaluados mostraron que el método IgG2 anti-PPD alcanzó la mayor especificidad de 95%, (CI: 88,3-101,8) con un valor predictivo positivo (VPP) de 75. La sensibilidad de los métodos estuvo entre 30% y 95%; dos métodos alcanzaron altas sensibilidades: los altos niveles de IFN-g en líquido pleural, con sensibilidad de 95% (CI: 85,5-104,5), con un valor predictivo negativo (VPN) de 97, seguido de los bajos niveles de IL-12p40 en suero, con una sensibilidad de 95% (CI: 85,5-104,5) con un VPN de 95,2. En contraste, ...

In vitro levels of cytokines in response to purified protein derivative (PPD) antigen in a population with high prevalence of pulmonary tuberculosis.

The control of mycobacterial infection by the host depends on cell-mediated immunity (CMI), involving activated macrophages, T cells, and type 1 cytokines (Th1). Here we evaluated the capacity of antigen-induced proliferation by peripheral blood mononuclear cells (PBMCs) and the production of proinflammatory tumor necrosis factor-alpha (TNF-α) and interleukin 12 (IL-12p40). The Th1 cytokine interferon-gamma (IFN-γ) and Th2 cytokines interleukin 4 (IL-4) and interleukin 5 (IL-5) in 62 pulmonary tuberculosis (TB) patients (40 Warao indigenous patients [WP] and 22 Creole non-indigenous patients [CP]) and 24 healthy controls (12 Warao indigenous controls [WC] and 12 Creole non-indigenous controls [CC]) at 24 and 48 hours in response to purified protein derivative (PPD) from Mycobacterium tuberculosis. The overall results revealed that testing of CP and CC' PBMCs for TNF-α, IFN-γ, and IL-12p40 production was higher compared with WP and WC' PBMCs after stimulating for 24 and 48 hours (p < 0.0001), within the WP group, the lower productions of IL-12p40 and IFN-γ significantly correlated (r(2) = 0.91, p < 0.01). Although in general there was interindividual variability in the observed responses of Th2 cytokines, especially with IL-4, there was a trend to produce higher PPD-induced IL-5 by WP' PBMCs compared with WC' PBMCs and CP' PBMCs at 24 and 48 hours, respectively. High IL-5 production correlated inversely with low IFN-γ production (r(2) = -0.97, p < 0.002). In conclusion, our results suggest that PPD-induced responses observed in patients from both populations can be divided into two groups: one group that activates Creole' PBMCs to preferentially secrete TNF-α, IL-12p40 and IFN-γ and another group that activates preferential secretion of IL-5 in Warao' PBMCs.

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.