Integrating systemic immune-inflammation index, fibrinogen, and T-SPOT.TB for precision distinction of active pulmonary tuberculosis in the era of mycobacterial disease research.

Background: The clinical challenge of differentiating suspected tuberculosis with positive T-SPOT.TB results persist. This study aims to investigate the utility of the Systemic Immune-Inflammation Index (SII), Fibrinogen, and T-SPOT.TB in distinguishing between active pulmonary tuberculosis (PTB) and non-tuberculous lung diseases. Methods: A retrospective analysis included 1,327 cases of active PTB with positive T-SPOT.TB results and 703 cases of non-tuberculous lung diseases from May 2016 to December 2020 at Meizhou People's Hospital. These were designated as the case group and the control group, respectively. The detection indicators of T-SPOT.TB: Early Secreted Antigenic Target 6 (ESAT-6), Culture Filtrate Protein 10 (CFP-10), as well as SII and Fibrinogen levels-were compared and analyzed for association and joint diagnostic value between the two groups. Results: The case group showed higher values of ESAT-6, CFP-10, SII, and Fibrinogen compared to the control group (all p < 0.001). In the case group, SII and Fibrinogen did not correlate with ESAT-6 and CFP-10 (∣rs∣ all < 0.3) but were positively correlated with C-reactive protein (CRP; rs all > 0.3). SII and Fibrinogen values in smear-positive pulmonary tuberculosis were higher than in smear-negative cases (all p < 0.05). The optimal diagnostic thresholds for ESAT-6, CFP-10, SII, and Fibrinogen in differentiating between active PTB and non-tuberculous lung diseases were 21.50 SFCs/106 PBMC, 22.50 SFCs/106 PBMC, 2128.32, and 5.02 g/L, respectively. Regression logistic analysis showed that ESAT-6 < 21.5 (OR: 1.637, 95% CI: 1.311-2.043, p < 0.001), CFP-10 < 22.5 (OR: 3.918, 95% CI: 3.138-4.892, p = 0.025), SII < 2128.32 (OR: 0.763, 95% CI: 0.603-0.967, p < 0.001), and FIB < 5.02 (OR: 2.287, 95% CI: 1.865-2.806, p < 0.001) were independent risk factors for active PTB. The specificity for ESAT-6 + CFP-10, ESAT-6 + CFP-10 + SII, ESAT-6 + CFP-10 + FIB, and ESAT-6 + CFP-10 + SII + FIB was 82.5%, 83.2%, 95.8%, and 80.1%, respectively, while sensitivity was 52.6%, 53.0%, 55.8%, and 44.7%, and positive predictive values were 85.0%, 85.6%, 84.1%, and 89.6%, respectively. Conclusion: SII and Fibrinogen are positively correlated with the degree of tuberculosis inflammation and the bacterial load of Mycobacterium tuberculosis. The combined detection of SII, Fibrinogen, and T-SPOT.TB is significant in distinguishing between active PTB with positive T-SPOT.TB results and non-tuberculous lung diseases.

Development and evaluation of a single-tube nested PCR with colorimetric assay for Mycobacterium tuberculosis detection.

Molecular techniques have revolutionized tuberculosis (TB) diagnosis by offering a faster and more sensitive approach, detecting Mycobacterium tuberculosis (Mtb) DNA directly from samples. Single-tube nested PCR (STNPCR) combines two PCR reactions with separate oligonucleotide sets in a single tube. Moreover, colorimetric methods in PCR products have been studied for pathogen detection. Thus, this study aimed to establish a novel system based on colorimetric STNPCR for Mtb detection using microtiter plates with IS6110-amplified fragments. The results showed a general colorimetric STNPCR detection limit of 1 pg/µl. Its general sensitivity and specificity were 76.62 and 60.53%, respectively, with kappa index agreement of 0.166.

Automatic bright-field smear microscopy for diagnosis of pulmonary tuberculosis.

In recent decades, many studies have been published on the use of automatic smear microscopy for diagnosing pulmonary tuberculosis (TB). Most of them deal with a preliminary step of the diagnosis, the bacilli detection, whereas sputum smear microscopy for diagnosis of pulmonary TB comprises detecting and reporting the number of bacilli found in at least 100 microscopic fields, according to the 5 grading scales (negative, scanty, 1+, 2+ and 3+) endorsed by the World Health Organization (WHO). Pulmonary TB diagnosis in bright-field smear microscopy, however, depends upon the attention of a trained and motivated technician, while the automated TB diagnosis requires little or no interpretation by a technician. As far as we know, this work proposes the first automatic method for pulmonary TB diagnosis in bright-field smear microscopy, according to the WHO recommendations. The proposed method comprises a semantic segmentation step, using a deep neural network, followed by a filtering step aiming to reduce the number of false positives (false bacilli): color and shape filtering. In semantic segmentation, different configurations of encoders are evaluated, using depth-wise separable convolution layers and channel attention mechanism. The proposed method was evaluated with a large, robust, and annotated image dataset designed for this purpose, consisting of 250 testing sets, 50 sets for each of the 5 TB diagnostic classes. The following performance metrics were obtained for automatic pulmonary TB diagnosis by smear microscopy: mean precision of 0.894, mean recall of 0.896, and mean F1-score of 0.895.

Diagnostic accuracy of tongue swab testing on two automated tuberculosis diagnostic platforms, Cepheid Xpert MTB/RIF Ultra and Molbio Truenat MTB Ultima.

Tongue dorsum swabbing is a potential alternative to sputum collection for tuberculosis (TB) testing. Previous studies showed that Cepheid Xpert MTB/RIF Ultra (Xpert Ultra) can detect Mycobacterium tuberculosis DNA on tongue swabs stored in buffer, with 72% sensitivity and 100% specificity relative to a sputum microbiological reference standard (sputum MRS). The present study evaluated a more convenient sample collection protocol (dry swab storage), combined with streamlined sample processing protocols, for evaluating two commercial TB diagnostic tests: Xpert Ultra and Molbio Truenat MTB Ultima (MTB Ultima). Copan FLOQSwabs were self-collected or collected by study workers from 321 participants in Western Cape, South Africa. All participants had symptoms suggestive of TB, and 245 of them had sputum MRS-confirmed TB (by sputum MGIT culture and/or Xpert Ultra). One tongue swab per participant was tested on Xpert Ultra, and another tongue swab was tested with MTB Ultima. Xpert Ultra was 75.5% sensitive and 100% specific relative to sputum MRS, similar to previous methods that used swabs stored in buffer. MTB Ultima was 71.6% sensitive and 96.9% specific relative to sputum MRS. When sample lysates that were false-negative or invalid by MTB Ultima were frozen, thawed, and re-tested, MTB Ultima sensitivity rose to 79.1%. Both tests were more sensitive with swabs from participants with higher sputum Xpert Ultra semi-quantitative results. Although additional development could improve diagnostic accuracy, these results further support tongue swabs as easy-to-collect samples for TB testing. IMPORTANCE: Tongue dorsum swabbing is a promising alternative to sputum collection for tuberculosis (TB) testing. Our results lend further support for tongue swabs as exceptionally easy-to-collect samples for high-throughput TB testing.

Explainable deep-neural-network supported scheme for tuberculosis detection from chest radiographs.

Chest radiographs are examined in typical clinical settings by competent physicians for tuberculosis diagnosis. However, this procedure is time consuming and subjective. Due to the growing usage of machine learning techniques in applied sciences, researchers have begun applying comparable concepts to medical diagnostics, such as tuberculosis screening. In the period of extremely deep neural nets which comprised of hundreds of convolution layers for feature extraction, we create a shallow-CNN for screening of TB condition from Chest X-rays so that the model is able to offer appropriate interpretation for right diagnosis. The suggested model consists of four convolution-maxpooling layers with various hyperparameters that were optimized for optimal performance using a Bayesian optimization technique. The model was reported with a peak classification accuracy, F1-score, sensitivity and specificity of 0.95. In addition, the receiver operating characteristic (ROC) curve for the proposed shallow-CNN showed a peak area under the curve value of 0.976. Moreover, we have employed class activation maps (CAM) and Local Interpretable Model-agnostic Explanations (LIME), explainer systems for assessing the transparency and explainability of the model in comparison to a state-of-the-art pre-trained neural net such as the DenseNet.

Design and development of multiepitope chimeric antigens by bioinformatic and bacterial based recombinant expression methods, with potential application for bovine tuberculosis serodiagnosis.

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8 M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis.

A bipolar intuitionistic fuzzy decision-making model for selection of effective diagnosis method of tuberculosis.

OBJECTIVES: Tuberculosis (TB) is a contagious illness caused by Mycobacterium tuberculosis. The initial symptoms of TB are similar to other respiratory illnesses, posing diagnostic challenges. Therefore, the primary goal of this study is to design a novel decision-support system under a bipolar intuitionistic fuzzy environment to examine an effective TB diagnosing method. METHODS: To achieve the aim, a novel fuzzy decision support system is derived by integrating PROMETHEE and ARAS techniques. This technique evaluates TB diagnostic methods under the bipolar intuitionistic fuzzy context. Moreover, the defuzzification algorithm is proposed to convert the bipolar intuitionistic fuzzy score into crisp score. RESULTS: The proposed method found that the sputum test (T3) is the most accurate in diagnosing TB. Additionally, comparative and sensitivity analyses are derived to show the proposed method's efficiency. CONCLUSION: The proposed bipolar intuitionistic fuzzy sets, combined with the PROMETHEE-ARAS techniques, proved to be a valuable tool for assessing effective TB diagnosing methods.

Higher Sensitivity of Xpert MTB/RIF Ultra Over Tuberculosis Culture for the Diagnosis of Spinal Tuberculosis With Open or Computed Tomography-Guided Biopsies.

Background: Diagnostic specimens for spinal tuberculosis (STB) are mostly collected via open surgery. Percutaneous computed tomography (CT)-guided biopsies are used in times of limited surgical availability. However, poor diagnostic accuracy of Mycobacterium tuberculosis (Mtb) culture has been reported with this method, due to limited sample volume and the paucibacillary nature of STB. We evaluated Xpert MTB/RIF Ultra on open and CT-guided biopsies as compared with the gold standard Mtb culture and histopathology. Methods: We conducted a prospective diagnostic accuracy study of Xpert Ultra, as compared with tuberculosis culture and histopathology, in adults with signs and symptoms of STB at a tertiary academic hospital in South Africa from November 2020 to December 2021. Diagnostic testing was performed on 31 patients with available samples. Results: Xpert Ultra had a sensitivity of 94.7% (95% CI, 75.3%-99.7%) and specificity of 100% (95% CI, 75.7%-100.0%) against a reference standard of Mtb culture and histopathology. Xpert Ultra had high diagnostic accuracy in open and CT-guided biopsy samples with sensitivity and specificity of 100% and 100% (open) and 89% and 100% (CT), respectively. Mtb culture had limited specificity for CT-guided biopsies (43%; 95% CI, 15.8%-74.9%). HIV-1 coinfection did not affect Mtb abundance measures by Xpert Ultra or culture. Xpert Ultra was also superior to culture for STB diagnosis in patients concurrently treated for pulmonary tuberculosis. Conclusions: Xpert Ultra detected more STB cases than culture for CT-guided biopsy samples. There was also no difference in sensitivity for open biopsies, irrespective of HIV-1 status, making it an important tool for rapid diagnosis, especially during times or in locations where open surgery is not possible or concurrent pulmonary tuberculosis treatment is initiated.

Bode Phase Angle Signaling of a TB Disease Biomarker.

Tuberculosis (TB) is a worldwide burden whose total control and eradication remains a challenge due to factors including false positive/negative diagnoses associated with the poor sensitivity of the current diagnostics in immune-compromised and post-vaccinated individuals. As these factors complicate both diagnosis and treatment, the early diagnosis of TB is of pivotal importance towards reaching the universal vision of a TB-free world. Here, an aptasensor for signaling an interferon gamma (IFN-γ) TB biomarker at low levels is reported. The aptasensor was assembled through gold-thiol interactions between poly(3,4-propylenedioxythiophene), gold nanoparticles, and a thiol-modified DNA aptamer specific to IFN-γ. The aptasensor sensitively detected IFN-γ in spiked pleural fluid samples with a detection limit of 0.09 pg/mL within a linear range from 0.2 pg/mL to 1.2 pg/mL. The good performance of the reported aptasensor indicates that it holds the potential for application in the early diagnosis of, in addition to TB, various diseases associated with IFN-γ release in clinical samples.

MXene-incorporated C60NPs and Au@Pt with dual-electric signal outputs for accurate detection of Mycobacterium tuberculosis ESAT-6 antigen.

Rapid and effective detection of Mycobacterium tuberculosis (MTB) is the crux of minimizing tuberculosis (TB) spread. Consequently, a new electrochemical aptasensor based on dual-signal output for ultrasensitive detection of MTB early secreted antigenic target 6 (ESAT-6) antigen was developed. Especially, a new nanocomposite MXene/C60NPs/Au@Pt was synthesized for signal generation and amplification. In this biosensing architecture, dual independent signal outputs were achieved by coupling the electrochemical redox activity of fullerene nanoparticles (C60NPs) with the effective electrocatalytic activity of Au@Pt nanoparticles. MXene possesses a large specific surface area, allowing densely loaded of these two electroactive materials, further improved sensing capability. In addition, specific ESAT-6 antigen binding aptamers were attached to Au@Pt to create the tracer label. With a typical sandwich format along with the introduction of the gold nanoparticle-loaded molybdenum disulfide (MoS2-Au) as the sensing interface, the limit of detection (LOD) of the proposed aptasensor was 2.88 fg mL-1 (DPV measurement) and 13.50 fg mL-1 (IT measurement), respectively, with a broad linear range of 100 fg mL-1 to 50 ng mL-1. Significantly, it exhibited better specificity and accuracy with a sensitivity of 97.5% and a specificity of 96.7% to distinguish healthy donors, other lung diseases and TB patients compared to commercial ELISA assay, holding a promising prospect in clinical diagnosis.

A flash signal amplification approach for ultrasensitive and rapid detection of single nucleotide polymorphisms in tuberculosis.

In recent years, the demand for rapid, sensitive, and simple methods for diagnosing deoxyribonucleic acid (DNA) has grown due to the increase in the variation of infectious diseases. This work aimed to develop a flash signal amplification method coupled with electrochemical detection for polymerase chain reaction (PCR)-free tuberculosis (TB) molecular diagnosis. We exploited the slightly miscible properties of butanol and water to instantly concentrate a capture probe DNA, a single-stranded mismatch DNA, and gold nanoparticles (AuNPs) to a small volume to reduce the diffusion and reaction time in the solution. In addition, the electrochemical signal was enhanced once two strands of DNA were hybridized and bound to the surface of the gold nanoparticle at an ultra-high density. To eliminate non-specific adsorption and identify mismatched DNA, the self-assembled monolayers (SAMs) and Muts proteins were sequentially modified on the working electrode. This sensitive and specific approach can detect as low as attomolar levels of DNA targets (18 aM) and is successfully applied to detecting tuberculosis-associated single nucleotide polymorphisms (SNPs) in synovial fluid. More importantly, as this biosensing strategy can amplify the signal in only a few seconds, it possesses a great potential for point-of-care and molecular diagnosis applications.

Rapid Identification of Lineage and Drug Resistance in Clinical Samples of Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis is a slow-growing bacterium, which could delay its diagnosis and, therefore, promote the spread of the disease. Whole-genome sequencing allows us to obtain the complete drug-resistance profile of the strain; however, bacterial cultivation of clinical samples, along with complex processing, is required. METHODS: In this work, we explore AmpliSeq, an amplicon-based enrichment method for preparing libraries for targeted next-generation sequencing, to identify lineage and drug resistance directly from clinical samples. RESULTS: In our study, 111 clinical samples were tested. The lineage was identified in 100% of the culture-derived samples (52/52), in 95% of the smear (BK)-positive clinical samples (38/40) and in 42.1% of the BK-negative clinical samples (8/19). The drug-resistance profile was accurately identified in all but 11 samples, in which some phenotypic and genotypic discrepancies were found. In this respect, our panels were not exact in the detection of streptomycin resistance for isolates derived from clinical samples, as an extremely high number of SNPs in the rrs and rrl genes were detected due to cross-contamination. CONCLUSION: This technique has demonstrated high sensitivity in obtaining the drug-resistance profile of the isolates, as even those samples with DNA concentrations below the detection limit of Qubit produced a result. AmpliSeq technology is cheaper than whole-genome sequencing, easy to perform by laboratory technicians and applicable to any microorganism using the Ion Torrent platform.

Pooling sputum samples for Xpert® MTB/RIF and Xpert® Ultra testing for TB diagnosis.

BACKGROUND: The use of molecular amplification as-says for TB diagnosis is limited by their costs and cartridge stocks. Pooling multiple samples to test them together is reported to have similar accuracy to individual testing and to save costs. METHODS: Two surveys of individuals with presumptive TB were conducted to assess the performance of pooled testing using Xpert® MTB/RIF (MTB/RIF) and Xpert® Ultra (Ultra). RESULTS: A total of 500 individuals were tested using MTB/RIF, with 72 (14.4%) being MTB-positive. The samples were tested in 125 pools, with 50 pools having ⩾1 MTB-positive and 75 only MTB-negative samples: 46/50 (92%, 95% CI 80.8-97.8) MTB-positive pools tested MTB-positive and 71/75 (94.7%, 95% CI 86.9-98.5) MTB-negative pools tested MTB-negative in the pooled test (agreement: 93.6%, κ = 0.867). Five hundred additional samples were tested using Ultra, with 60 (12%) being MTB-positive. Samples were tested in 125 pools, with 42 having ⩾1 MTB-positive and 83 only MTB-negative samples: 35/42 (83.6%, 95% CI 68.6-93.0) MTB-positive pools tested MTB-positive and 82/83 (98.8%, 95% CI 93.5-100.0) MTB-negative pools tested MTB-negative in the pooled test (agreement: 93.6%, κ = 0.851; P > 0.1 between individual and pooled testing). Pooled testing saved 35% (MTB/RIF) and 46% (Ultra) of cartridges. CONCLUSIONS: Pooled and individual testing has a high level of agreement and improves testing efficiency.

CONTEXTE: Le coût et les stocks de cartouches des tests d'amplification moléculaire limitent leur utilisation pour le diagnostic de la TB. Regrouper plusieurs échantillons afin de les tester en même temps aurait une précision similaire à celle des tests individuels et permettrait de réaliser des économies. MÉTHODES: Deux enquêtes ont été menées auprès de personnes avec une TB présumée afin d'évaluer la performance des tests groupés en utilisant le test Xpert® MTB/RIF (MTB/RIF) et le test Xpert® Ultra (Ultra). RÉSULTATS: Au total, 500 personnes ont été testées par test MTB/RIF, dont 72 (14,4%) étaient MTB-positives. Les échantillons ont été testés dans 125 groupes, dont 50 groupes avaient ⩾1 échantillons MTB-positifs et 75 uniquement des échantillons MTB-négatifs : 46/50 (92% ; IC 95% 80,8­97,8) groupes MTB-positifs ont été testés MTB-positifs et 71/75 (94,7% ; IC 95% 86,9­98,5) groupes MTB-négatifs ont été testés MTB-négatifs dans le test groupé (concordance : 93,6% ; κ = 0,867). Cinq cents échantillons supplémentaires ont été testés par test Ultra, dont 60 (12%) étaient MTB-positifs. Les échantillons ont été testés dans 125 groupes, dont 42 avaient ⩾1 échantillons MTB-positifs et 83 uniquement des échantillons MTB-négatifs : 35/42 (83,6% ; IC 95% 68,6­93,0) groupes MTB-positifs ont été testés MTB-positifs et 82/83 (98,8% ; IC 95% 93,5­100,0) groupes MTB-négatifs ont été testés MTB-négatifs dans le test groupé (concordance : 93,6% ; κ = 0,851 ; P > 0,1 entre les tests individuels et groupés). Les tests groupés ont permis d'économiser 35% (MTB/RIF) et 46% (Ultra) des cartouches. CONCLUSIONS: Les tests groupés et individuels présentent un niveau élevé de concordance et améliorent l'efficacité des tests.

Relationship between ambient temperature at sampling and the interferon gamma test result for bovine tuberculosis in cattle.

Bovine tuberculosis (bTB) is a disease of significant economic and zoonotic importance, therefore, optimising tests for the identification of Mycobacterium bovis infected cattle is essential. The Interferon Gamma (IFN-γ) Release Assay (IGRA) can diagnose M. bovis infected cattle at an early stage, is easy to perform and can be used alongside skin tests for confirmatory purposes or to increase diagnostic sensitivity. It is known that IGRA performance is sensitive to environmental conditions under which samples are taken and transported. In this study, the association between the ambient temperature on the day of bleeding and the subsequent IGRA result for bTB was quantified using field samples from Northern Ireland (NI). Results of 106,434 IGRA results (2013-2018) were associated with temperature data extracted from weather stations near tested cattle herds. Model dependent variables were the levels of IFN-γ triggered by avian purified protein derivative (PPDa), M. bovis PPD (PPDb), their difference (PPD(b-a)) as well as the final binary outcome (positive or negative for M. bovis infection). IFN-γ levels after both PPDa and PPDb stimulation were lowest at the extremes of the temperature distribution for NI. The highest IGRA positive probability (above 6%) was found on days with moderate maximum temperatures (6-16 °C) or moderate minimum temperatures (4-7 °C). Adjustment for covariates did not lead to major changes in the model estimates. These data suggest that IGRA performance can be affected when samples are taken at high or low temperatures. Whilst it is difficult to exclude physiological factors, the data nonetheless supports the temperature control of samples from bleeding through to laboratory to help mitigate post-collection confounders.

Sensitive Urine Immunoassay for Visualization of Lipoarabinomannan for Noninvasive Tuberculosis Diagnosis.

Lipoarabinomannan (LAM) is a prospective noninvasive biomarker for tuberculosis (TB) diagnosis. Here, we report a visual immunoassay of high sensitivity for detecting LAM in urine samples toward TB diagnosis. This method uses a DNA-linked immunosorbent of LAM, followed by a transduction cascade into amplified visual signals using quantum dots (QDs) and calcein reaction with Cu2+ and copper nanoparticles (Cu NPs). The limit of detection (LOD) for LAM in the urine reaches 2.5 fg/mL and 25 fg/mL using a fluorometer and length readouts on strips, respectively, demonstrating an ultrahigh sensitivity. The clinical validation of the proposed assay was performed with 147 HIV-negative clinical urine specimens. The results show the sensitivity of test is 94.1% (16/17) for confirmed TB (culture-positive) and 85% (51/60) for unconfirmed TB (clinical diagnosis without positive culture results), respectively, when the test cutoff value is 40 fg/mL for TB. Its specificity is 89.2% (25/28) in non-TB and nontuberculous mycobacterial patients. The area under the curve (AUC) was 0.86 when controls were non-TB and LTBI patients, while the AUC was 0.92 when controls were only non-TB patients. This highly sensitive visual immunoassay of LAM has shown potential for noninvasive diagnosis of TB using urine samples.

A multiplex PCR assay for the differentiation of Mycobacterium tuberculosis complex reveals high rates of mixed-lineage tuberculosis infections among patients in Ghana.

In low-resource settings with high tuberculosis (TB) burdens, lack of rapid diagnostic methods for detection and differentiation of Mycobacterium tuberculosis complex (MTBC) is a major challenge affecting TB management. This study utilized comparative genomic analyses of MTBC lineages; M. tuberculosis, M. africanum Lineages 5/6 and M. bovis to identify lineage-specific genes. Primers were designed for the development of a Multiplex PCR assay which was successful in differentiating the MTBC lineages. There was no cross-reaction with other respiratory pathogens tested. Validation of the assay using clinical samples was performed with sputum DNA extracts from 341 clinically confirmed active TB patients. It was observed that 24.9% of cases were caused by M. tuberculosis, while M. africanum L5 & L6 reported 9.0% and 14.4%, respectively. M. bovis infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. This multiplex PCR assay will allow speciation of MTBC lineages in low-resource regions, providing rapid differentiation of TB infections to select appropriate medication at the earliest possible time point. It will also be useful in epidemiological surveillance studies providing reliable information on the prevalence of TB lineages as well as identifying difficult to treat cases of mixed-lineage tuberculosis infections.

Tuberculosis extrapulmonar: un problema de salud pública.

Extrapulmonary tuberculosis is defined as that case of tuberculosis clinically diagnosed and confirmed by bacteriological studies that affects tissues and organs outside the lung parenchyma. Mexico is in third place among Latin American countries in terms of the incidence of pulmonary and extrapulmonary tuberculosis. Culture methods are still the gold standard for the diagnosis of extrapulmonary tuberculosis since they identify the species and susceptibility to drugs.

La tuberculosis extrapulmonar es aquella tuberculosis diagnosticada clínicamente y confirmada por estudios bacteriológicos que afecta a tejidos y órganos fuera del parénquima pulmonar. México es el tercer lugar en América Latina en incidencia de tuberculosis pulmonar y extrapulmonar. Los métodos de cultivo siguen siendo el método de referencia para el diagnóstico de tuberculosis extrapulmonar, ya que identifican la especie y la sensibilidad a los fármacos.

Serum biomarkers in patients with unilateral or bilateral active pulmonary tuberculosis: Immunological networks and promising diagnostic applications.

The present observational study was designed to characterize the integrative profile of serum soluble mediators to describe the immunological networks associated with clinical findings and identify putative biomarkers for diagnosis and prognosis of active tuberculosis. The study population comprises 163 volunteers, including 84 patients with active pulmonary tuberculosis/(TB), and 79 controls/(C). Soluble mediators were measured by multiplexed assay. Data analysis demonstrated that the levels of CCL3, CCL5, CXCL10, IL-1ß, IL-6, IFN-γ, IL-1Ra, IL-4, IL-10, PDGF, VEGF, G-CSF, IL-7 were increased in TB as compared to C. Patients with bilateral pulmonary involvement/(TB-BI) exhibited higher levels of CXCL8, IL-6 and TNF with distinct biomarker signatures (CCL11, CCL2, TNF and IL-10) as compared to patients with unilateral infiltrates/(TB-UNI). Analysis of biomarker networks based in correlation power graph demonstrated small number of strong connections in TB and TB-BI. The search for biomarkers with relevant implications to understand the pathogenetic mechanisms and useful as complementary diagnosis tool of active TB pointed out the excellent performance of single analysis of IL-6 or CXCL10 and the stepwise combination of IL-6 â†’ CXCL10 (Accuracy = 84 %; 80 % and 88 %, respectively). Together, our finding demonstrated that immunological networks of serum soluble biomarkers in TB patients differ according to the unilateral or bilateral pulmonary involvement and may have relevant implications to understand the pathogenetic mechanisms involved in the clinical outcome of Mtb infection.

Performance of IS6110-LAMP assay for detection of Mycobacterium tuberculosis complex in blood and urine samples from patients with extrapulmonary tuberculosis.

BACKGROUND: The diagnosis of extrapulmonary tuberculosis (EPTB) shows numerous difficulties because of non-specific symptomatology and low sensitivity of conventional methods. Loop-mediated isothermal amplification (LAMP) is a fast and low-cost technique, which can amplify under isothermal conditions an amount of target DNA copies into approximately a billion copies. OBJECTIVE: The present study aimed to evaluate a IS6110-LAMP system for Mycobacterium tuberculosis detection in blood and urine samples from patients with EPTB. METHODS: The collected samples (n = 122) were stratified in two groups: Group EPTB - patient samples with confirmed EPTB (n = 61); Group non-TB - patient samples without TB (n = 61). The urine samples underwent decontamination, and the components of blood samples were separated (plasma and PBMC). DNA extractions were performed in all biological samples followed by IS6110-LAMP assay technique. The detection limit was evaluated through dilution curves (1:10) using Mtb reference strain (H37Rv) genomic DNA. FINDINGS: The detection limit of IS6110-LAMP was 10 fg/µL (∼10-20 bacilli/µL). The IS6110-LAMP technique sensitivity and specificity were 95.65 % and 79.25 %, respectively, with a general kappa agreement index of 0.762. MAIN CONCLUSIONS: Based on these results, IS6110-LAMP test showed considerable diagnostic parameters, being able to aid in the speed and accuracy of the final EPTB diagnosis.