Heat shock proteins: Possible biomarkers in pulmonary and extrapulmonary tuberculosis

Tuberculosis (TB) and Tuberculous meningitis (TBM) caused by Mycobacterium tuberculosis (MTB) continue to be a major cause of morbidity and mortality. Therefore there is a need to explore potential biomarkers and heat shock proteins [Hsp(s)] could be one such candidate. We found that host (Hsp 25, Hsp 60, Hsp 70 and Hsp 90) and MTB Hsp(s) (Hsp 16, Hsp 65 and Hsp 71) to be an important feature of the immune response in human clinical samples of pulmonary and extrapulmonary TB patients and in MTB infected monocytes. Notably, the host (Hsp 25, Hsp 70 and Hsp 90) and MTB (Hsp 16, Hsp 65 and Hsp 71) Hsp(s) increases significantly in the clinical samples as well as in cell line model after TB infection. Collectively, results revealed that alteration in immune response leads to a change in the both host and MTB Hsp profile, highlighting them as possible biomarkers for the disease.     

Improved Case Detection Using Xpert Mycobacterium tuberculosis/Rifampicin Assay in Skeletal Tuberculosis

Background: In India, musculoskeletal tuberculosis (TB) accounts for 10%–25% of extrapulmonary TB. Data on drug-resistant skeletal TB are lacking. At present, the diagnosis is based mainly on radiological techniques. Laboratory confirmation of skeletal TB is delayed as 6–8 weeks are required for culture results. Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay is a fully automated test which simultaneously detects MTB and RIF resistance within 3 h. Hence, this study was done to compare the yield of case detection using Xpert assay in comparison with culture in specimens received from clinically suspected skeletal TB cases. Methods: Retrospective analysis of microscopy, culture and Xpert assay results was carried out on specimens received in laboratory from skeletal TB cases from January 2016 to December 2017. Results: Of the 201 patients analysed, majority of the specimens were obtained from the spine (55.72%). MTB was detected in 48.68% of tissue and 24% of pus specimens. Xpert assay was detected MTB in 67 (33.33%) specimens of which 53 (47.32%) were from the spine. Culture was detected MTB in 66 (32.83%) specimens. Xpert assay was detected two specimens more than culture. One specimen was positive by only culture. RIF-resistant MTB was detected in 10 (14.92%) specimens by Xpert assay. Conclusion: The spine is the most common site involved. Tissue specimen is better for early diagnosis. High RIF resistance in skeletal TB is an alarming situation. Ability of Xpert MTB/RIF assay for rapid and simultaneous detection of MTB and RIF resistance in comparison with culture makes it a useful diagnostic tool in skeletal TB.     

Evaluation of a semi-automated Seegene PCR workflow with MTB,MDR, and NTM detection for rapid screening of tuberculosis in a low-prevalence setting

In areas of low tuberculosis (TB) prevalence, laboratory diagnosis of TB may essentially cover non-tuberculous mycobacteria (NTM) in addition to Mycobacterium tuberculosis (MTB). In this study, a semi-automated PCR workflow distinguishing MTB and NTM (Anyplex™ MTB/NTMe, Seegene) and subsequently detecting MTB isoniazid/rifampicin resistance (Allplex™ MTB/MDRe, Seegene) was evaluated for replacing smear microscopy of acid-fast bacilli as the rapid screening method for TB. With 279 clinical samples, 47 cultures positive for MTB and 76 for NTM, the Anyplex™ MTB/NTMe assay and smear microscopy showed equal sensitivities (49.6% vs 50.8%, respectively) but Anyplex™ MTB/NTMe was more sensitive for MTB (63.8% vs 25.6%) than for NTM (40.8% vs 64.5%). Allplex™ MTB/MDRe showed a slightly higher sensitivity of 68.1% for MTB (32/47 positive, n = 222). Antibiotic resistance profiles were correctly identified for all MTB isolates (one MDR isolate). Specificity was 100% for both assays. Anyplex™ MTB/NTMe detected all the 18 NTM species present in the study. The analytical performance of the evaluated high-throughput workflow was relatively weak compared to culture but potentially adequate as a rapid screening method analogous to smear microscopy with additional differentiation between TB, MDR-TB, and NTM.     

Performance of a commercial nucleic acid amplification test with extrapulmonary specimens for the diagnosis of tuberculosis

The laboratory diagnosis of tuberculosis (TB) on extrapulmonary specimens is particularly challenging. A number of commercial nucleic acid amplification tests able to detect and identify Mycobacterium tuberculosis (MTB) complex directly from respiratory secretions have been developed, but their use on extrapulmonary samples still calls for validation. The BDProbeTec ET Mycobacterium tuberculosis Complex Direct Detection Assay (DTB) was applied to 918 consecutive extrapulmonary specimens (collected from 863 patients), including 84 gastric aspirates, 145 urine, 136 sterile body fluids, 83 cerebrospinal (CSF) fluids, 237 fine-needle aspirates, 175 pus, 56 biopsies, and two stool specimens. The results were compared with those of acid-fast staining and culture (solid plus liquid media), setting the combination of culture and clinical diagnosis as the gold standard. Ninety-two specimens yielded culture positive for MTB and 24 (smear- and culture-negative) were from patients with TB clinical diagnosis. Of these, 96 were DTB-positive, including all of those from culture-negative TB cases. From 26 specimens, nontuberculous mycobacteria were grown. Two of these specimens were positive by the DTB assay. Finally, of the 776 samples that were smear- and culture-negative for acid-fast bacilli (AFB), collected from patients for whom the diagnosis of TB was excluded, six were DTB-positive. The overall sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of extrapulmonary samples were 82.7, 99.0, 92.3, and 97.8%, respectively. Although, at present, amplification assays cannot replace culture techniques, DTB proved to be rapid and specific for the detection of MTB in extrapulmonary samples.     

Evaluation of the Xpert MTB/RIF test accuracy for diagnosis of tuberculosis in areas with a moderate tuberculosis burden

The Xpert MTB/RIF assay (Xpert) is a molecular assay used for direct detection of Mycobacterium tuberculosis (MTB) in clinical specimens. In this study, we aimed to assess the accuracy of the Xpert assay for the diagnosis of tuberculosis (TB) in TB suspected patients from the northern region of Iran. The obtained results were compared with the culture method. The sputum specimens were examined using the Xpert assay, smear microscopy, and solid culture media as a reference diagnostic tool. Among 293 presumptive TB cases, 92 (31.4%) were positive according to the culture method. The Xpert method detected 88 (95.7%) cases that were positive according to the culture method, compared with 78 (84.8%) positive cases according to smear microscopy. The overall sensitivity and specificity of the Xpert method for TB diagnosis were 95.7% and 99%, respectively. Also, the sensitivity and specificity for smear microscopy were 84.8% and 97.5%, respectively. The Xpert assay showed high overall sensitivity and specificity; thus, it can be effectively used for the early and accurate diagnosis of MTB in TB endemic areas. In addition, the agreement between semi‐quantitative results of Xpert and smear microscopy assays could be helpful in evaluating transmission potential in TB patients.     

Immunogenicity and cross-reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> of proteoliposomes derived from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB.     

Progress in serodiagnosis of Mycobacterium tuberculosis infection

One-third of the world population is estimated to have Mycobacterium tuberculosis infection. Accurate and timely identification of infected individuals is critical for treatment and control. The current diagnostic methods lack the desired sensitivity and specificity, require sophisticated equipment and skilled workforce or take weeks to yield results. Diagnosis of extrapulmonary TB, TB-HIV co-infection, childhood TB and sputum smear-negative pulmonary TB pose serious challenges. Interest in developing serodiagnostic methods is increasing because detection of antibody is rapid, simple and relatively inexpensive, and does not require a living cell for detection. Three types of tests, namely screening tests to overcome diagnostic delay, specific tests for diagnosis of extrapulmonary TB and other bacteriologically negative cases, and tests for vaccine-induced immunity need critical consideration. Several factors must be considered to develop serodiagnostic methods for TB. Antigen recognition by infected individuals is highly heterogeneous due to stage of disease, differences in HLA types, strain of the bacilli, health of the patient and bacillary load. With advances in molecular biological techniques, a number of novel antigens have been identified. Some of these antigens have proven valuable in detecting specific antibodies in some of the most challenging TB patients. The best example is a fusion protein containing several M. tuberculosis proteins (e.g. CFP-10, MTB8, MTB48, MTB81 and the 38-kDa protein) which showed encouraging results in detecting antibodies in sera of patients, including TB-HIV co-infection. This review presents progress made in the serodiagnosis of TB during the last decade.     

Diagnostic Accuracy of Xpert MTB/RIF Assay in Extrapulmonary Tuberculosis

Introduction: The WHO endorsed Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay, has been evaluated for pulmonary TB in a number of studies but very few have investigated it for extrapulmonary specimens. The present study evaluates the performance of Xpert MTB/RIF assay in the diagnosis of extrapulmonary TB (EPTB). Aim and Objectives: The aim of the study is to determine sensitivity and specificity of Xpert MTB/RIF assay for diagnosis of EPTB and RIF resistance in comparison to culture on Lowenstein–Jensen (LJ) medium and proportion method (PM), respectively. Materials and Methods: A total of 738 specimens from clinically suspected cases of EPTB were subjected to Ziehl–Neelsen staining, Xpert MTB/RIF assay and culture on LJ medium. PM was done on MTB isolates. Results: The sensitivity, specificity of Xpert MTB/RIF assay for diagnosis of EPTB were 84.91% (95% confidence interval [CI] 72.41%–93.25%) and 86.72% (95% CI 83.94%–89.17%) and for RIF resistance detection were 60.00% (95% CI 32.29%–83.66%) and 94.74% (95% CI 73.97%–99.87%), respectively. Among culture-positive cases, the sensitivity of Xpert MTB/RIF assay was 94.12% in smear positive and 80.56% in smear-negative cases. Xpert MTB/RIF showed maximum sensitivity of MTB detection from lymph node specimens (100% [95% CI 54.07%–100.00%]) and other body fluids (100% [95% CI 15.81%–100.00%]). Conclusion: The present study establishes Xpert MTB/RIF assay as a promising tool in the rapid diagnosis of EPTB and detection of RIF resistance.     

Use of Transrenal DNA for the Diagnosis of Extrapulmonary Tuberculosis in Children: a Case of Tubercular Otitis Media

The diagnosis of tuberculosis (TB) is difficult in children, especially for smear-negative pulmonary and extrapulmonary TB, which are common at this age. We report an 11-year-old girl with TB otitis media with negative smear microscopy and Xpert MTB/RIF but positive Mycobacterium tuberculosis-specific transrenal DNA (Tr-MTB-DNA) test results and culture for M. tuberculosis.     

Modified TB rapid test by proteinase K for rapid diagnosis of pleural tuberculosis

The diagnosis of pleural tuberculosis continues to be a challenge due to the low sensitivity of traditional diagnostic methods. Better and more rapid tests are needed for diagnosis of pleural TB. In this study, pleural fluids were tested with rapid test to determine Mycobacterium tuberculosis (MTB antigen). Affinity chromatography was used to purify specific polyclonal antibodies against MTB antigen. Pleural samples after decontamination were treated with proteinase K. Rapid test for pleural fluids was prepared by specific antibody. Rapid test was performed on 85 pleural fluid patients. The patients had a mean age of 46.55 ± 15.96 years and 38 were men. The performance of rapid test, using proteinase K, was found to be the most impressive: sensitivity 93%, specificity 94%, PPV 90%, and NPV 96% compared with adenosine deaminase test (ADA), PCR, smear, and culture. The present study did demonstrate that modified TB rapid test can substantially improve the diagnosis of extrapulmonary TB.     

Purification,biochemical characterization and immunogenicity of SA5K,a secretion antigen of Mycobacterium tuberculosis

Mycobacterium tuberculosis (MTB) secretory proteins are generally considered important antigens for immune protection against tuberculosis (TB). An 8.3-kDa secretory antigen of MTB and Mycobacterium bovis bacillus Calmette-Guérin (BCG), called SA5K, was recently identified and cloned in our laboratory. In this report, recombinant SA5K containing a histidine hexamer was expressed in Escherichia coli and purified to investigate its biochemical structure and to establish whether it was immunogenic for healthy sensitized and nonsensitized human donors and for patients infected with MTB. The protein nucleotide sequence was shown to be identical in BCG and in MTB. SA5K revealed an abnormal electrophoretic mobility in SDS-PAGE that made it look lighter than it is in Western blotting. While recombinant SA5K was poorly recognized by T lymphocytes from patients with pulmonary TB, it elicited proliferation of CD4+ T lymphocytes in the vast majority of healthy individuals sensitized to mycobacterial antigens by BCG vaccination. At a serum dilution of 1 : 80, antibodies reacting against recombinant SA5K were found in 67% of sera from TB patients and in 73% of sera from healthy subjects. The percentage of positive subjects dropped at higher serum dilutions, but no significant difference in the recognition rate was observed between TB patients and healthy donors and between healthy vaccinated and nonvaccinated subjects. Owing to the high percentage of sera from healthy subjects who recognized SA5K in Western blotting, the antigen seems to exhibit, at least in the present form, a poor specificity for an employment for a serodiagnosis of TB.     

Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens

The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance.     

Xpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis: a diagnostic evaluation study

ObjectivesThe diagnosis of extrapulmonary tuberculosis (EPTB) is often made on clinical suspicion alone, resulting in both under- and overdiagnosis and relatively poor outcomes. In this study, we evaluated the clinical utility of the Xpert MTB/RIF on routinely collected extrapulmonary specimens in Ethiopia.MethodsThis study was carried out at Jimma University Specialized Hospital, Southwest Ethiopia. Extrapulmonary specimens were collected from 572 patients clinically suspected of suffering from EPTB. All specimens were tested for TB by smear microscopy, culture, and Xpert MTB/RIF. The diagnostic accuracy of Xpert MTB/RIF was calculated and compared to a composite reference standard (CRS), comprising clinical and laboratory results.ResultsIn total, 572 extrapulmonary specimens (279 lymph node, 159 pleural, 80 peritoneal, 45 cerebrospinal, and nine pericardial fluids) were tested. The pooled sensitivity and specificity of Xpert MTB/RIF were calculated to be 75% (95% CI 70–80) and 98% (95% CI 97–100) respectively when compared to the CRS. The highest sensitivity was documented for lymph node specimens (90%; 95% CI 86–94), moderate sensitivity for cerebrospinal fluid (53%; 95% CI 28–79), while the sensitivity was lowest for pleural (30%; 95% CI 17–44) and peritoneal (32%; 95% CI 12–51) fluids. Xpert MTB/RIF in addition detected rifampicin resistance in 13 patients, in perfect agreement with results from the line probe assay.ConclusionsXpert MTB/RIF may be used as initial diagnostic tool for testing of lymph node specimens from patients suspected of having TB lymphadenitis. The added value of Xpert MTB/RIF to diagnose pleural or peritoneal TB is limited by its poor sensitivity.     

PCR-膜芯片检测石蜡包埋组织中结核杆菌耐药基因突变

目的探讨PCR-膜芯片技术检测石蜡包埋组织中结核杆菌耐药基因突变的可行性及其在临床病理中的应用价值。方法采集100例石蜡包埋的肺或淋巴结组织标本(临床诊断为结核患者80例,非结核患者20例),TB膜芯片检测标本中的结核杆菌IS6110基因,阳性者利用12对特异性引物进行多重PCR,扩增产物与含有59个探针的耐药-TB膜芯片反向点杂交检测结核杆菌耐药基因突变并测序验证。结果 TB膜芯片检测石蜡包埋组织标本中的结核杆菌IS6110基因,以临床诊断为标准时,灵敏度为52.5%(42/80),特异度为100%(20/20);以抗酸染色结果为标准时,灵敏度为90.5%(19/21),特异度为61.0%(36/59)。耐药-TB膜芯片检测石蜡包埋组织结核杆菌耐药基因突变,在42例结核杆菌IS6110基因阳性的标本中,检出INH基因突变2例,RFP或SM基因突变各1例,INH、RFP和EMB基因同时突变1例,与测序结果基本符合,提示上述5例可能为耐药结核病。结论应用PCR-膜芯片技术可提高石蜡包埋组织中结核杆菌基因的检出率,耐药-TB膜芯片检测结果可为耐药结核病的病理学诊断提供有价值的参考。     

Humoral Immune Responses against the Mycobacterium tuberculosis 38-Kilodalton,MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis

The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging. The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in 250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and 73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.The control of tuberculosis (TB) remains challenging in China (18). Currently, the diagnosis of active TB mainly relies on clinical symptoms, radiologic findings, and the detection of Mycobacterium tuberculosis in clinical samples using smear staining and mycobacterial culture. However, the diagnosis of TB in smear- and culture-negative TB patients is difficult. The detection of M. tuberculosis-specific antibodies in human sera has been an important aid in diagnosis of TB. Notably, several antigens have been demonstrated to have merit in TB diagnosis, including the 38-kDa protein, which is commonly used in serodiagnostic tests (4, 5, 8, 13, 19, 22, 23). Previous studies suggest that the antibody responses to M. tuberculosis antigens are heterogeneous among individuals (17) so that the detection of antibodies against a single antigen usually has a low sensitivity for diagnosis of TB, especially for bacterium-negative cases. Therefore, it may be valuable to evaluate antibodies against the 38-kDa antigen and other major antigens for the diagnosis of active TB (14, 15).Notably, the MTB48, CFP-10 (culture filtrate protein 10), and ESAT-6 (6-kDa early secreted antigen target of M. tuberculosis) genes are conserved in M. tuberculosis and Mycobacterium bovis isolates but partially deleted or absent in M. bovis BCG as well as in most nontuberculous mycobacteria (NTM) (1-3, 10, 16). Importantly, the proteins encoded by these genes are immunogenic (7, 9, 12, 16). In this study, we cloned the 38-kDa, MTB48, CFP-10, and ESAT-6 genes and generated recombinant 38-kDa, MTB48, and CFP-10/ESAT-6 fusion proteins in Escherichia coli. Subsequently, we developed an enzyme-linked immunosorbent assay (ELISA) for the characterization of serum antibodies against 38-kDa, MTB48, and CFP-10/ESAT-6 antigens in a population of 250 active TB patients and 260 healthy subjects. We found that characterization of antibodies against multiple M. tuberculosis antigens were valuable for the diagnosis of active TB.     

Ex vivo responses for interferon-gamma and proinflammatory cytokine secretion to low-molecular-weight antigen MTB12 of Mycobacterium tuberculosis during human tuberculosis

MTB12 protein, also called CFP-2, is a major and early secreted component of Mycobacterium tuberculosis. However, its role during mycobacterial infection has been poorly characterized. In this study, we purified the native MTB12 protein and investigated the profile of MTB12-induced cytokines [interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-6], in early tuberculosis (TB) patients (n = 20) and healthy controls (n = 35). The cytokine profiles were compared with those induced by the 30-kDa antigen (Ag). In healthy controls, MTB12-induced IFN-gamma production was markedly decreased in peripheral blood mononuclear cells compared with 30-kDa Ag-induced IFN-gamma. In TB patients, the mean IFN-gamma level induced by MTB12 was lower than that induced by the 30-kDa Ag, albeit the difference was not significant. After 2 months of anti-TB therapy, both the MTB12- and 30-kDa-induced IFN-gamma levels were significantly increased in TB patients. MTB12-induced TNF-alpha and IL-6 levels were prominently upregulated in monocyte-derived macrophages from TB patients, but they were not significantly different from those induced by the 30-kDa Ag. Further, the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase was required for the induction of TNF-alpha and IL-6 by MTB12, as well as by the 30-kDa Ag. Collectively, these data suggest that the MTB12 protein plays an essential role for proinflammatory responses through the MAPK pathway during the early stages of human TB, even though its T-cell immunoreactivity is weaker than that of the 30-kDa Ag.     

Evaluation of two tuberculosis PCR assays for routine use in a clinical setting of low population and low tuberculosis prevalence

Today, there are numerous different molecular diagnostic assays for the detection of tuberculosis (TB), allowing the optimization of rapid detection of TB according to the clinical need. In this study, two high‐throughput TB PCR assays with combined antimicrobial resistance detection, Anyplex? II MTB/MDR (Seegene) and RealTime MTB + RealTime MTB RIF/INH Resistance (Abbott Molecular), were evaluated for routine use in a clinical setting of low population and low TB prevalence in Finland. The RealTime MTB assay was 100% concordant (22/22 positive, n = 169) with the reference methods (culture and Xpert MTB/RIF PCR assay, Cepheid). However, with a limitation of four separate PCR cycles per kit, the routine use in a low TB‐prevalence setting would easily lead to wasting most of the RIF/INH Resistance reagents. The Anyplex? II MTB/MDR assay usability was more adaptive to suit the clinical setting but the assay sensitivity was considerably lower (86%, 19/22 positive, n = 76) being closer to the sensitivity of smear microscopy. The findings of this study suggest that the evaluated high‐throughput MTB/MDR assays are evidently suboptimal for routine use in a low population, low TB‐prevalence setting. In addition, neither of the two assays covers non‐tuberculous mycobacteria and could therefore not fully replace acid‐fast staining as the initial screening method.     

Mucosal delivery of antigen‐coated nanoparticles to lungs confers protective immunity against tuberculosis infection in mice

Mucosal boosting of BCG‐immunised individuals with a subunit tuberculosis (TB) vaccine would be highly desirable, considering that the lungs are the principal port of entry for Mycobacterium tuberculosis (MTB) and the site of the primary infection and reactivation. However, the main roadblock for subunit TB vaccine development is the lack of suitable adjuvants that could induce robust local and systemic immune responses. Here, we describe a novel vaccine delivery system that was designed to mimic, in part, the MTB pathogen itself. The surface of yellow carnauba wax nanoparticles was coated with the highly immunogenic Ag85B Ag of MTB and they were directed to the alveolar epithelial surfaces by the incorporation of the heparin‐binding hemagglutinin adhesion (HBHA) protein. Our results showed that the i.n. immunisation of BCG‐primed BALB/c mice with nanoparticles adsorbed with Ag85B‐HBHA (Nano‐AH vaccine) induced robust humoral and cellular immune responses and IFN‐γ production, and multifunctional CD4⁺ T cells expressing IFN‐γ, IL‐2 and TNF‐α. Mice challenged with H37Rv MTB had a significantly reduced bacterial load in their lungs when compared with controls immunised with BCG alone. We therefore conclude that this immunisation approach is an effective means of boosting the BCG‐induced anti‐TB immunity.