Active Tuberculosis Patients Research Articles

Stealth Adapted Viruses with Genetically Unstable Rhesus Monkey Cellular Sequences A Possible Forerunner of Complex Human Illnesses

Real time PCR is a widely used for molecular diagnosis of tuberculosis in routine diagnosis laboratory. There are many commercially avaliable system. The sensitivity of the system varies according to DNA extraction from the sample and target specific primers and probes and commercial systems. In this study, the sensitivity and specificity of AdvanSure TB/NTM real time PCR kit were evaluated for detecting mycobacteria in smear positive and negative sputum specimens of 35 active pulmonary tuberculosis patients. All specimens were corrected using EZN staining, solid (Löwenstein Jensen) and liquid culture (MGIT). Culture and clinical findings were used as gold standards in the evaluation of all PCR results. Overall true positivity was found 25 ( 71.42%). When smear negative samples were evaluated together with culture results, only 3 patients were found positive by PCR test in 10 samples (30%).

NOTCH1 and DLL4 are involved in the human tuberculosis progression and immune response activation

Tuberculosis (TB) is the leading cause of mortality among infectious diseases worldwide. The study of molecular targets for therapy and diagnosis suggested that Notch signaling is an important pathway for the maintenance of the immune response during Mycobacterium tuberculosis (Mtb) infection. We evaluated the participation of the Notch pathway in the modulation of immune response during Mtb infection, and observed that patients with active TB had increased DLL4 expression in intermediate and non-classic monocytes. Further, patients with moderate and advanced lung injury have higher Notch1 expression in CD4+ T cells when compared to patients with a minimal lung injury. When we considered the severity of disease in active TB patients, the expression of the DLL4 in intermediate monocytes and the expression of Notch1 in CD4+ T cells are positively correlated with the degree of lung injury. In vitro, PBMCs treated with the Notch pharmacological inhibitor reduced the production of IL-17A and IL-2, whereas anti-hDLL4 treatment promoted a significant increase in TNF-α and phagocytosis. We suggest that Notch1 and DLL4 are associated with immune response activation in human tuberculosis, and can be a novel target to be exploited in the future in the searching of biomarkers.

Decreased Expression of CD69 on T Cells in Tuberculosis Infection Resisters.

BackgroundCD69 is a biomarker of T-cell activation status, but its activation status in human Mycobacterium tuberculosis (Mtb) infection remains elusive.MethodsA set of cohorts of patients with different tuberculosis (TB) infection status including active TB patients (ATB), latent tuberculous infection patients (LTBI) and close contacts (CCs) of ATB was designed, and the expression profiles of CD69 and several T-cell markers were determined on Mtb antigen-stimulated T cells by flow cytometry.ResultsThe frequencies of CD4+ and CD8+ T cells were both comparable among Mtb-infected individuals including ATB and LTBI, which guaranteed the consistency of the background level. A t-Distributed Stochastic Neighbor Embedding (tSNE) analysis on a panel of six phenotypic markers showed a unique color map axis gated on T cells in the CCs group compared with ATB and LTBI populations. By further gating on cells positive for each individual marker and then overlaying those events on top of the tSNE plots, their distribution suggested that some markers were expressed differently in the CCs group. Further analysis showed that the expression levels of CD69 on both CD4+ and CD8+ T cells were significantly lower in the CCs group, especially in interferon-γ-responding T cells.ConclusionOur findings suggest that the T-cell activation status of CD69 is associated with Mtb infection and may have the potential to distinguish LTBI from those populations who have been exposed continuously to Mtb but have not become infected.

QuantiFERON-TB Gold Plus combined with HBHA-Induced IFN-γ release assay improves the accuracy of identifying tuberculosis disease status

ObjectiveQFT-Plus's newly added antigen elicited a specific CD8 T-cell response, which is closely related to active TB (ATB), and the IGRA based on Heparin-binding haemagglutinin (HBHA-IGRA) is a promising tool in latent tuberculosis infection (LTBI) diagnosis. The objective of our study is to evaluate whether the combination of QFT-Plus and HBHA-IGRA can improve the diagnosis accuracy of ATB and LTBI. Methods135 healthcare workers (HCWs) and 57 patients with active pulmonary TB in a Chinese TB Hospital were recruited, HCWs underwent screening for LTBI using the QFT-Plus assay. Flow cytometry was used to analyze the distribution of peripheral blood T lymphocyte subsets in active TB patients with positive culture result. Then, the patients with ATB, individuals with LTBI and healthy TB-uninfected controls (HC) were tested by QFT-Plus and HBHA-IGRA respectively, and the efficiency of distinguishing LTBI from ATB by QFT-Plus and HBHA-IGRA were evaluated by Receiver Operating Characteristic (ROC) curves. ResultsQFT-Plus TB2-TB1 which was positively correlated with CD8 T-cell response (r = 0.731, P < 0.001) in peripheral blood was significantly higher in ATB than LTBI and HC (median 0.47 IU/mL versus 0.02 IU/mL and 0.00 IU/mL, respectively; both P < 0.0001). While the HBHA-induced IFN-γ response did not differ between ATB and HC (median 12.12 pg/mL versus 10.95 pg/mL; P = 0.463), but was significantly higher in the LTBI (median 69.67 pg/mL; both P < 0.0001). The ROC area under the curve (AUC) for identifying ATB and LTBI was 0.769 (95% CI: 0.652–0.886; P = 0.0001) for QFT-Plus TB2-TB1, and 0.886 (95% CI:0.791–0.982; P＜0.0001) for HBHA-IGRA. After combining the HBHA-IGRA with QFT-Plus results, the accuracy of identifying ATB and LTBI was improved to 85.7% from 74.3%. ConclusionsHBHA-based IGRA better differentiates between LTBI and ATB compared to QFT-Plus CD8 T-cell response. In addition, combining HBHA-IGRA and QFT-Plus improves the accuracy of identifying tuberculosis disease status.

Diagnostic benefits of adding EspC, EspF and Rv2348-B to the QuantiFERON Gold In-tube antigen combination

Interferon (IFN)-γ release assays (IGRAs) are used to diagnose latent tuberculosis (TB) infection (LTBI). To improve the accuracy of these tests, different approaches, such as alternative cytokine detection and using different antigens, are considered. Following this purpose, this study aims to evaluate the addition of EspC, EspF and Rv2348-B to those present in the QuantiFERON-TB Gold In-Tube (QFN-G-IT). We included 115 subjects: 74 active TB patients, 17 LTBI individuals and 24 healthy controls. Whole blood samples were collected in QFN-G-IT and in-house tubes containing different combinations of EspC, EspF and Rv2348-B, together with ESAT-6, CFP-10, and TB7.7. After overnight incubation at 37 ºC, plasma was harvested and IFN-γ quantified. IFN-γ levels in the QFN-G-IT and in-house tubes correlated very good (Spearman Rho(r) > 0.86). In-house antigen combinations distinguished healthy individuals from those with active TB and LTBI (specificities and sensitivities higher than 87.5% and 96.3%, respectively [AUC > 0.938]). Adding EspC, EspF and Rv2348-B, increased the sensitivity of the test, being the addition of EspC and Rv2348-B the combination that yielded a higher sensitivity with no specificity loss. Addition of these antigens could improve diagnosis in patients with impaired or immature immune response who are at high risk of developing TB.

Characterization of antibody response against 16kD and 38kD of M. tuberculosis in the assisted diagnosis of active pulmonary tuberculosis.

BackgroundIn view of the inability of traditional etiological methods to diagnose pulmonary tuberculosis rapidly and effectively, the antibody responses against 38kD and 16kD-antigens of Mycobacterium tuberculosis (M. tuberculosis) were both detected in order to obtain a better serological detection method for M. tuberculosis.MethodsM. tuberculosis-secreted protein 38kD and membrane protein 16kD were prokaryotically expressed and purified, and then used as detection antigens. A novel evolved immunoglobulin-binding molecule (NEIBM)-ELISA method was used to detect antibody levels against 38kD and 16kD in active tuberculosis patients (confirmed tuberculosis cases and clinically diagnosed cases), to explore the significance of these two antigens in serological detection of M. tuberculosis, and to study the diagnostic value of the combined detection of the two antigens in active pulmonary tuberculosis.ResultsThe results showed that the positive detection rates of the 16kD antigen and 38kD antigen of M. tuberculosis were higher (about 44%) in the confirmed cases of tuberculosis, and there was no significant difference in the positive detection rates of the two antigens (P=0.786). The combined detection of these two antigens showed that the positive detection rate could be increased to 61.5%, which was significantly better than the detection effect of the two antigens alone. The positive detection rates of 16kD and 38kD antigens were 26–30% in clinically diagnosed tuberculosis cases, which were lower than those in confirmed tuberculosis cases, and there was no significant difference in the positive detection rates of the two antigens (P=0.242). The detection effect of the two combined antigens was better than that of the 16kD and 38kD antigens alone, but the detection rate was still lower than that of the confirmed tuberculosis cases.ConclusionsThis study found that the detection effect of 16kD and 38kD antigens was similar in confirmed cases and clinically diagnosed cases of pulmonary tuberculosis, and that the detection effect needs to be further improved. The combined detection of the two antigens showed a significantly better detection effect than the two antigens alone, suggesting that the combined detection of multiple antigens can be used for serological diagnosis of M. tuberculosis infection in clinic.

The single recombinant M. tuberculosis protein DPPD provides enhanced performance of skin testing among HIV-infected tuberculosis patients

Diagnostic testing for M. tuberculosis infection has advanced with QuantiFERON and GeneXpert, but simple cost-effective alternatives for widespread TB screening has remained elusive and purified protein derivative (PPD)-based tuberculin skin testing (TST) remains the most widely used method. PPD-based tests have reduced performance, however, in BCG vaccinees and in individuals with immune deficiencies. We compared the performance of skin testing with the recombinant DPPD protein against that of a standard PPD-based skin test. Our data indicates similar performance of DPPD and PPD (r2 = 0.7689) among HIV-negative, active TB patients, all of whom presented greater than 10 mm induration following administration. In contrast to results demonstrating that PPD induced indurations greater than 5 mm (i.e., the recommended threshold for positive results in this population) in only half (19 of 38) of the HIV positive TB patients, 89.5% (34 of 38) of these participants developed indurations greater than 5 mm when challenged with DPPD. Importantly, none of the patients that were positive following PPD administration were negative following DPPD administration, indicating markedly improved sensitivity of DPPD among HIV-infected individuals. Our data indicate that DPPD has superior performance in skin testing than the current TST standard.

Identification of candidate host serum and saliva biomarkers for a better diagnosis of active and latent tuberculosis infection.

In our work, we aim to identify new candidate host biomarkers to discriminate between active TB patients (n = 28), latent infection (LTBI; n = 27) and uninfected (NoTBI; n = 42) individuals. For that, active TB patients and their contacts were recruited that donated serum and saliva samples. A multiplex assay was performed to study the concentration of different cytokines, chemokines and growth factors. Proteins with significant differences between groups were selected and logistic regression and the area under the ROC curve (AUC) was used to assess the diagnostic accuracy. The best marker combinations that discriminate active TB from NoTBI contacts were [IP-10 + IL-7] in serum and [Fractalkine + IP-10 + IL-1α + VEGF] in saliva. Best discrimination between active TB and LTBI was achieved using [IP-10 + BCA-1] in serum (AUC = 0.83) and IP-10 in saliva (p = 0.0007; AUC = 0.78). The levels of TNFα (p = 0.003; AUC = 0.73) in serum and the combination of [Fractalkine+IL-12p40] (AUC = 0.83) in saliva, were able to differentiate between NoTBI and LTBI contacts. In conclusion, different individual and combined protein markers could help to discriminate between active TB and both uninfected and latently-infected contacts. The most promising ones include [IP-10 + IL-7], [IP-10 + BCA-1] and TNFα in serum and [Fractalkine + IP-10 + IL-1α + VEGF], IP-10 and [Fractalkine+IL-12p40] in saliva.

Diagnostic performance of culture filtered protein 10-specific perforin in pediatric patients with active tuberculosis.

Background Mycobacterium tuberculosis (Mtb)‐specific perforin were significantly increased in patients with tuberculosis. This study aims to evaluate the diagnosis value of Mtb‐specific perforin in pediatric patients with tuberculosis.MethodsDiagnostic performance of perforin levels induced by 6‐kDa early secreted antigen target (ESAT6) or culture filtered protein 10 (CFP10) were evaluated in eighty‐six samples from children participants by receiver operating characteristic curve analysis. Flow cytometry was used to detect the expression of perforin and INF‐γ of CD4+, CD8+ T cells in response to CFP10 stimulation.ResultsAfter ex vivo stimulation, levels of ESAT6/CFP10‐specific perforin in LTBI patients were significantly higher than active TB (ATB) patients, non‐tuberculosis infection (non‐TB), and health control (HC) individuals. The diagnostic efficacy of CFP10‐specific perforin for TB diagnosis was significantly higher than ESAT6‐specific perforin and T‐SPOT assay, and when 0.74 ng/mL was taken as the cutoff value, the sensitivity, specificity, and accuracy were 97.83%, 87.5%, and 93.02%. CFP10‐specific perforin in both CD4+ and CD8+ T cells were significantly higher in ATB patients compared to HCs and further increased in LTBI patients. However, INF‐γ was mainly secreted by CD4+ T cells and showed no significant difference between LTBI and ATB patients. In addition, CFP10‐specific perforin can effectively distinguish between ATB and LTBI with the cutoff value of 1.80 ng/mL. Sensitivity and specificity were 88.46% and 85.62%, respectively.ConclusionsCFP10‐specific perforin may be used as a novel cellular immunity‐based diagnostic marker of pediatric patients with tuberculosis, and with the potential for discriminating ATB from LTBI.

IFN-Gamma and IL-2 Secretion after ESAT-6-CFP-10 (EC-610) Fusion Antigen Stimulation from Patients with Active Lung Tuberculosis and Latent Lung Tuberculosis

The Secretion of IFN-Gamma and IL-2 After ESAT-6-CFP-10 Fusion Antigen Stimulation in Active and Latent TB Patients. This study held to discover how immune responses work and to know the pathogenesis of active TB and latent TB patients. This study used PBMC to stimulate T Cells with ESAT-6 and CFP-10 antigen fusion, and measure the level of IFN-gamma and IL-2 with ELISA antibody sandwich (U-Cytech). 16 ml of blood were drawn to 5 tubes. ESAT-6 CFP-20 inducted one tube with QuantiFERON for IFN-gamma assay. The other four tubes were PBMC isolated using Ficoll-Paque, and pre-incubated with stimulation of ESAT-6 CFP-10 fusion antigen for 24-72 hours at 370 C and measured using T-Spot and ELISA reader. We got from this study that there are no significant differences in IFN-gamma levels for both groups with active TB and latent TB. Measurement of IL-2 levels showed significant differences between the two group.

Mycobacterium tuberculosis-specific CD4 T cells expressing CD153 inversely associate with bacterial load and disease severity in human tuberculosis

Recent data from mice and non-human primate models of tuberculosis suggested that CD153, a TNF super family member, plays an important role in Mycobacterium tuberculosis (Mtb) control. However, this molecule has not been comprehensively evaluated in humans. Here, we show that the proportion of Mtb-specific CD4 T cells expressing CD153 was significantly reduced in active TB patients compared to latently infected persons. Importantly, the CD153+ Mtb-specific CD4 response inversely correlated with lung bacterial load, inferred by Xpert cycle threshold, irrespective of HIV status. Antitubercular treatment partially restored CD153 expression on Mtb-specific CD4 T cells. This is the first report of a subset of Mtb-specific CD4 T cells showing strong negative correlation with bacterial burden. Building on substantial evidence from animal models implicating CD153 as a mediator of host protection, our findings suggest it may play a similar role in humans and its measurement may be useful to evaluate TB vaccine efficacy.

Amerindian genetic ancestry as a risk factor for tuberculosis in an amazonian population.

In recent years, the incidence of tuberculosis (TB) has declined worldwide, although this disease still occurs at relatively high rates in Amerindian populations. This suggests that the genetic ancestry of Amerindians may be an important factor in the development of infections, and may account for at least some of the variation in infection rates in the different populations. The present study investigated the potential influence of Amerindian genetic ancestry on susceptibility to tuberculosis in an Amazon population. The study included 280 patients diagnosed with tuberculosis and 138 asymptomatic hospital employees with no history of TB, but who were in contact with bacterially active TB patients. Ancestry analysis was run on a set of 61 Ancestry-Informative Markers to estimate European, African, and Amerindian genetic ancestry using STRUCTURE v2.2. The TB group had significantly higher Amerindian ancestry in comparison with the control group, and significantly lower European ancestry. Amerindian ancestry in the 20–60% range was found to be the principal risk factor for increased susceptibility to TB. The results of the study indicate that Amerindian ancestry is an important risk factor for susceptibility to TB in the admixed population of the Brazilian Amazon region.

Evolution of Interferon-Gamma Release Assay Results and Submillisievert Chest CT Findings among Close Contacts of Active Pulmonary Tuberculosis Patients.

BackgroundLatent tuberculosis (TB) infection among TB contacts is diagnosed using plain chest radiography and interferon-gamma release assays (IGRAs). However, plain chest radiographs often miss active TB, and the results of IGRA could fluctuate over time. The purpose of this study was to elucidate changes in the results of the serial IGRAs and in the findings of the serial submillisievert chest computed tomography (CT) scans among the close contacts of active pulmonary TB patients.MethodsPatients age 20 or older with active pulmonary TB and their close contacts were invited to participate in this study. Two types of IGRA (QuantiFERON-TB Gold In-Tube assay [QFT-GIT] and the T-SPOT.TB test [T-SPOT]) and submillisievert chest CT scanning were performed at baseline and at 3 and 12 months after enrollment.ResultsIn total, 19 close contacts participated in this study. One was diagnosed with active pulmonary TB and was excluded from further analysis. At baseline, four of 18 contacts (22.2%) showed positive results for QFT-GIT and T-SPOT; there were no discordant results. During the follow-up, transient and permanent positive or negative conversions and discordant results between the two types of IGRAs were observed in some patients. Among the 17 contacts who underwent submillisievert chest CT scanning, calcified nodules were identified in seven (41.2%), noncalcified nodules in 14 (82.4%), and bronchiectasis in four (23.5%). Some nodules disappeared over time.ConclusionThe results of the QFT-GIT and T-SPOT assays and the CT images may change during 1 year of observation of close contacts of the active TB patients.

Evaluation of Advansure TB/NTM Real Time PCR Kit for Detection of Mycobacteria in Sputum Specimens

Real time PCR is a widely used for molecular diagnosis of tuberculosis in routine diagnosis laboratory. There are many commercially avaliable system. The sensitivity of the system varies according to DNA extraction from the sample and target specific primers and probes and commercial systems. In this study, the sensitivity and specificity of AdvanSure TB/NTM real time PCR kit were evaluated for detecting mycobacteria in smear positive and negative sputum specimens of 35 active pulmonary tuberculosis patients. All specimens were corrected using EZN staining, solid (Löwenstein Jensen) and liquid culture (MGIT). Culture and clinical findings were used as gold standards in the evaluation of all PCR results. Overall true positivity was found 25 ( 71.42%). When smear negative samples were evaluated together with culture results, only 3 patients were found positive by PCR test in 10 samples (30%).

Developing and verifying automatic detection of active pulmonary tuberculosis from multi-slice spiral CT images based on deep learning

Diagnosis of tuberculosis (TB) in multi-slice spiral computed tomography (CT) images is a difficult task in many TB prevalent locations in which experienced radiologists are lacking. To address this difficulty, we develop an automated detection system based on artificial intelligence (AI) in this study to simplify the diagnostic process of active tuberculosis (ATB) and improve the diagnostic accuracy using CT images. A CT image dataset of 846 patients is retrospectively collected from a large teaching hospital. The gold standard for ATB patients is sputum smear, and the gold standard for normal and pneumonia patients is the CT report result. The dataset is divided into independent training and testing data subsets. The training data contains 337 ATB, 110 pneumonia, and 120 normal cases, while the testing data contains 139 ATB, 40 pneumonia, and 100 normal cases, respectively. A U-Net deep learning algorithm was applied for automatic detection and segmentation of ATB lesions. Image processing methods are then applied to CT layers diagnosed as ATB lesions by U-Net, which can detect potentially misdiagnosed layers, and can turn 2D ATB lesions into 3D lesions based on consecutive U-Net annotations. Finally, independent test data is used to evaluate the performance of the developed AI tool. For an independent test, the AI tool yields an AUC value of 0.980. Accuracy, sensitivity, specificity, positive predictive value, and negative predictive value are 0.968, 0.964, 0.971, 0.971, and 0.964, respectively, which shows that the AI tool performs well for detection of ATB and differential diagnosis of non-ATB (i.e. pneumonia and normal cases). An AI tool for automatic detection of ATB in chest CT is successfully developed in this study. The AI tool can accurately detect ATB patients, and distinguish between ATB and non- ATB cases, which simplifies the diagnosis process and lays a solid foundation for the next step of AI in CT diagnosis of ATB in clinical application.

The spatial analysis of extrapulmonary tuberculosis spreading and its interactions with pulmonary tuberculosis in Samarinda, East Kalimantan, Indonesia.

BackgroundExtrapulmonary Tubercolosis (EPTB) is an infectious disease that affects tissue outside the lungs. EPTB patients cannot be source of infection, therefore the findings in the community indicate that there are still active pulmonary TB patients acting as a source of infection. Understanding distributions of EPTB can be used as indicator to individuate the unmonitored source of TB transmission in the community.ObjectivesThe aim of this study is to analyze EPTB using spatial modeling based on patients’ location.MethodsThis study is an observational research with spatial analysis approach using SatScanv.9.4.4 and ArcGis v.10.4. Involving 46 samples of EPTB patients in Anatomy Pathology Laboratory of RSUD Abdul Wahab Sjahranie in 2017 and 7 pulmonary TB patients who were contacts of EPTB patients. The distribution of EPTB patients is mostly located in areas with high population density.ResultsThe results showed that the distribution pattern of EPTB patients was mostly in areas with high population densities. Space-time permutation model shows there are 3 clusters of EPTB with a 2.91, 0.97, 1.13 km radius centered on -0.504177 S/117.092132 E, -0.476895 S /117.141700 E, -0.517031 S/117.092132 E.ConclusionThe distribution of patients with EPTB and pulmonary TB indicates there is an interaction between EPTB and pulmonary TB in the cluster area. Bernoulli model shows that there is 1 cluster of EPTB and pulmonary TB with relative risk 5.29, radius of 3.19 km, and centered on - 0.458159 S / 117.149945 E.

Impact of Intermediate Hyperglycemia and Diabetes on Immune Dysfunction in Tuberculosis.

BackgroundPeople with diabetes have an increased risk of developing active tuberculosis (TB) and are more likely to have poor TB-treatment outcomes, which may impact on control of TB as the prevalence of diabetes is increasing worldwide. Blood transcriptomes are altered in patients with active TB relative to healthy individuals. The effects of diabetes and intermediate hyperglycemia (IH) on this transcriptomic signature were investigated to enhance understanding of immunological susceptibility in diabetes-TB comorbidity.MethodsWhole blood samples were collected from active TB patients with diabetes (glycated hemoglobin [HbA1c] ≥6.5%) or IH (HbA1c = 5.7% to <6.5%), TB-only patients, and healthy controls in 4 countries: South Africa, Romania, Indonesia, and Peru. Differential blood gene expression was determined by RNA-seq (n = 249).ResultsDiabetes increased the magnitude of gene expression change in the host transcriptome in TB, notably showing an increase in genes associated with innate inflammatory and decrease in adaptive immune responses. Strikingly, patients with IH and TB exhibited blood transcriptomes much more similar to patients with diabetes-TB than to patients with only TB. Both diabetes-TB and IH-TB patients had a decreased type I interferon response relative to TB-only patients.ConclusionsComorbidity in individuals with both TB and diabetes is associated with altered transcriptomes, with an expected enhanced inflammation in the presence of both conditions, but also reduced type I interferon responses in comorbid patients, suggesting an unexpected uncoupling of the TB transcriptome phenotype. These immunological dysfunctions are also present in individuals with IH, showing that altered immunity to TB may also be present in this group. The TB disease outcomes in individuals with IH diagnosed with TB should be investigated further.

Enhanced tenacity of mycobacterial aerosols from necrotic neutrophils

The tuberculosis agent Mycobacterium tuberculosis is primarily transmitted through air, but little is known about the tenacity of mycobacterium-containing aerosols derived from either suspensions or infected neutrophils. Analysis of mycobacterial aerosol particles generated from bacterial suspensions revealed an average aerodynamic diameter and mass density that may allow distant airborne transmission. The volume and mass of mycobacterial aerosol particles increased with elevated relative humidity. To more closely mimic aerosol formation that occurs in active TB patients, aerosols from mycobacterium-infected neutrophils were analysed. Mycobacterium-infected intact neutrophils showed a smaller particle size distribution and lower viability than free mycobacteria. In contrast, mycobacterium-infected necrotic neutrophils, predominant in M. tuberculosis infection, revealed particle sizes and viability rates similar to those found for free mycobacteria, but in addition, larger aggregates of viable mycobacteria were observed. Therefore, mycobacteria are shielded from environmental stresses in multibacillary aggregates generated from necrotic neutrophils, which allows improved tenacity but emphasizes short distance transmission between close contacts.

Changes in T-lymphocyte subsets and risk factors in human immunodeficiency virus-negative patients with active tuberculosis

PurposeImmune function imbalance is closely associated with the occurrence and development of infectious diseases. We studied the characteristics of changes in T-lymphocyte subsets and their risk factors in HIV-negative patients with active tuberculosis (ATB).MethodsT-lymphocyte subsets in 275 HIV-negative ATB patients were quantitatively analyzed and compared with an Mycobacteriumtuberculosis-free control group. Single-factor and multifactor analyses of clinical and laboratory characteristics of patients were also conducted.ResultsIn ATB patients, CD4 and CD8 T-cell counts decreased, and the levels were positively interrelated (r = 0.655, P < 0.0001). After 4 weeks of antituberculosis treatment, CD4 and CD8 T-cell counts increased significantly but remained lower than in the control group. CD4 and CD8 cell counts were negatively associated with the extent of lesions detected in the chest by computed tomography (all P < 0.05). Although not reflected in the CD4/CD8 ratio, CD4 and CD8 cell counts differed between drug-resistant TB patients and drug-susceptible TB patients (P = 0.030). The multivariate analysis showed prealbumin, alpha-1 globulin, body mass index, and platelet count were independent risk factors for decreased CD4 cell count (all P < 0.05), while age and platelet count were independent risk factors for decreased CD8 cell count (all P < 0.05).ConclusionCD4 and CD8 T-cell counts showed the evident value in predicting ATB severity. An increase in the CD4/CD8 ratio may be a critical clue of drug resistance in ATB. Although the factors influencing CD4 and CD8 are not identical, our results indicated the importance of serum protein and platelets to ATB patients’ immune function.

Reliable naked-eye detection of Mycobacterium tuberculosis antigen 85B using gold and copper nanoshell-enhanced immunoblotting techniques

A straightforward and naked-eye–sensitive nanoshell-enhanced immunoblotting technique was successfully developed to detect Mycobacterium tuberculosis antigen 85B (Ag85B). The immunoblotting assay included the intensification of the signal of the gold or copper nanoshell on the surface of gold-binding polypeptide fused to 50B14 (GBP-50B14) antibody-conjugated gold nanoparticles (AuNPs). The recombinant GBP-50B14 fusion antibody, which has gold-binding and antigen-binding abilities, shows high affinity toward the AuNP surface as well as Ag85B. The formation of a gold nanoshell or copper nanoshell on the surface of AuNPs leads to the generation of a purple or red spot that can be observed with the naked eye. The gold- and copper-enhanced intensity corresponds to the concentration of Ag85B and provides a detection limit of 0.93 and 0.21 ng/mL by a phone camera and 1.56 and 0.75 ng/mL by the naked eye, respectively. Owing to the higher analytical performance of copper nanoshell enrichment, this immunoblotting technique was successfully applied to determine Ag85B in clinical urine samples. Using this nanoplatform to determine the complexity of Ag85B and CFP10 tuberculosis antigens, the strong reactivity of urine samples from active tuberculosis patients showed 71.4% sensitivity and 86.7% specificity, suggesting excellent application potential in nanomedicine to reliably detect tuberculosis biomarkers for the accurate diagnosis of active Tuberculosis.