Tuberculosis Assay Research Articles

Assessing and screening for T-cell epitopes from Mycobacterium tuberculosis RD2 proteins for the diagnosis of active tuberculosis

The Region of D eletion 2 (RD2) of Mycobacterium tuberculosis encodes reserved antigens that contribute to bacterial virulence. Among these antigens, Rv1983, Rv1986, Rv1987, and Rv1989c have been shown to be immunodominant in infected cattle; however, their diagnostic utility has not been evaluated in humans.In this study, we screened 87 overlapping synthetic peptides encoded by five RD2 proteins for diagnosing tuberculosis epitopes in 50 active tuberculosis (TB) cases, 31 non-tuberculosis patients and 36 healthy individuals. A pool of promising epitopes was then assessed for their diagnostic value in 233 suspected TB patients using a whole blood IFN-γ release assay.Only 10 peptides were recognized by more than 10% of active tuberculosis patients. The IFN-γ release responses to Rv1986-P9, P15, P16, Rv1988-P4, P11, and Rv1987-P11 were significantly higher in the active TB group than in the control groups (p<0.05). The whole blood IFN-γ release assay based on these epitopes yielded a sensitivity of 51% and a specificity of 85% in diagnosing active tuberculosis, and the corresponding results using the T-SPOT.TB assay were 76% and 75%, respectively.In conclusion, these results suggest that the six epitopes from the RD2 of M. tuberculosis have potential diagnostic value in TB.

Challenges of TB and HIV co-treatment: updates and insights.

In the past few years, tuberculosis (TB) has overtaken HIV as the infectious disease with the highest global mortality. Successful management of this syndemic will require improved diagnostic tests, shorter preventive therapies, and more effective treatments, particularly in light of drug-resistant TB. Results from several major studies have been published or presented recently, including the development of a more sensitive rapid, molecular assay for TB; several new symptom-based screening tools; use of a 1-month regimen for TB prevention; the results of early vs. delayed TB preventive therapy for pregnant women; newer drugs and regimens for multidrug-resistant tuberculosis; and pharmacokinetic, safety, and efficacy studies of new HIV drugs in combination with TB treatment. We reviewed each of these topic areas and summarize relevant findings for the management of TB and HIV co-infection. Moving forward, as new treatment regimes for HIV or TB are developed, consideration of the HIV-TB co-infected patient must figure prominently, both when determining the diagnostic tests employed and to assess properly the drug-drug and drug-disease interactions that influence dosing, safety, and response.

Novel High Sensitivity Tuberculosis Point-of-Care Test for People Living with HIV

Background: Most tuberculosis-related deaths in people living with HIV (PLHIV) could be prevented with earlier diagnosis and treatment. The only commercially available tuberculosis point-of-care test (Alere DetermineTM TB LAM Ag (AlereLAM)) has suboptimal sensitivity which restricts its use in clinical practice. The novel Fujifilm SILVAMP TB LAM (FujiLAM) test was designed to achieve superior diagnostic accuracy. Methods: We performed a blinded assessment of FujiLAM on biobanked urine samples from three independent prospective cohort studies of hospitalized PLHIV at two South African district hospitals. Diagnostic accuracy was determined against both microbiological (MRS) and composite reference standards (CRS, including clinical diagnoses), in comparison with AlereLAM. Findings: A total of 968 newly admitted PLHIV were included. The prevalence of microbiologically-confirmed TB was 62% and the median CD4 count 86 cells/µl. In a meta-analysis, the estimated sensitivity of the FujiLAM was superior at 70·4% (95%CI: 53·0-83·1) over that of AlereLAM at 42·3% (31·7-51·8), against the MRS, with a difference of 28.1% (21.5-34.4). Specificities were 90·8% (86·0-94·4) and 95·0% (87·7-98·8) for the FujiLAM and AlereLAM against the MRS, respectively, with a non-significant difference of -4.2% (-12.7-4.4). Against the CRS, specificities of both assays were higher (FujiLAM 95·7% and AlereLAM 98·2%) but sensitivities were lower (FujiLAM 64·9% and AlereLAM 38·2%). In the subgroup of patients with CD4 ≤100 cells/µl, the sensitivity of FujiLAM was 84·2% (71·4-91·4) versus 57·3% (42·2-69·6) for AlereLAM. Interpretation: In comparison to AlereLAM, FujiLAM offers superior diagnostic sensitivity, while maintaining specificity, and has the potential to transform rapid point-of-care tuberculosis diagnosis for hospitalized PLHIV. FujiLAM's suitability for settings of intended use needs to be assessed in a prospective evaluation. Funding: GHIT Fund, UK DFID, Dutch MoFA, BMGF, Australian DFAT, German BMBF through KfW, Wellcome Trust, DST/ NRF of South Africa, and South African MRC. Declaration of Interest: TB, EI, AM, SO, CCB, and CMD are 384 employed by FIND. FIND is a not for-profit foundation that supports the evaluation of publicly prioritized tuberculosis assays and the implementation of WHO-approved (guidance and prequalification) assays using donor grants. FIND has product evaluation agreements with several private sector companies that design diagnostics for tuberculosis and other diseases. These agreements strictly define FIND’s independence and neutrality vis-a-vis the companies whose products get evaluated and describe roles and responsibilities. AP and TB report patents in the field of lipoarabinomannan detection. All other authors declare no competing interests. Ethical Approval: All study-related activities were approved by the Human Research Ethics Committee (HREC) of the University of Cape Town (UCT). Written informed consent was obtained from patients, as per study protocols. Study participation did not affect standard of care. Reporting followed STARD guidelines.12 167 Retrospective urine LAM testing was supervised by the sponsor, FIND, and was performed at UCT in April 2018.

Active tuberculosis in patients with systemic lupus erythematosus from Southern China: a retrospective study.

To investigate the characteristics and associated factors for Mycobacterium tuberculosis (TB) infection in patients with systemic lupus erythematosus (SLE) from Southern China. A retrospective study of 1108 patients admitted to the First Affiliated Hospital of Sun Yat-Sen University from January 2007 to December 2017 was performed. Demographic and clinical characteristics, laboratory data, and radiographic manifestations were recorded. A total of 59 (5.3%) lupus patients with active TB were included. Pulmonary TB occurred in 41 (69.5%) patients. Single lobe involvement was showed in 14 (34.1%) patients. Multi-lobar involvement, including miliary TB (36.6%), was presented in 27 (65.8%) patients. Lower lobe involvement accounted for 31 (75.6%) of the cases. Extrapulmonary TB occurred in 18 (30.5%) patients. Nearly one-third (35.6%) of the patients developed disseminated TB. T-SPOT.TB assay was performed in 23 patients and positive in 18 patients (78.3%). Nineteen patients (32.2%) had co-infection with TB and other pathogens, most of which were bacterial-associated (52.6%). Lymphopenia was predominant in TB-infected patients, especially in those with disseminated TB. Multivariate logistic regression analysis found that lymphopenia [odds ratio (OR) = 2.19, 95% confidence interval (CI) 1.03-4.63, P = 0.04] and the accumulated doses of glucocorticoid (GC) (OR = 2.32, 95% CI 1.69-3.20, P < 0.001) were associated with TB. TB infection is a common comorbidity in patients with SLE. Manifestations of pulmonary computed tomography (CT) scan are relatively atypical. Co-infection with TB and other pathogens is not rare. Lymphopenia and the accumulated doses of GC are associated with TB infection in lupus patients.

Xpert® MTB/RIF assay for extrapulmonary tuberculosis and rifampicin resistance.

Background Tuberculosis (TB) is the world’s leading infectious cause of death. Extrapulmonary TB accounts for 15% of TB cases, but the proportion is increasing, and over half a million people were newly diagnosed with rifampicin‐resistant TB in 2016. Xpert® MTB/RIF (Xpert) is a World Health Organization (WHO)‐recommended, rapid, automated, nucleic acid amplification assay that is used widely for simultaneous detection of Mycobacterium tuberculosis complex and rifampicin resistance in sputum specimens. This Cochrane Review assessed the accuracy of Xpert in extrapulmonary specimens.

Diagnostic accuracy of the loop-mediated isothermal amplification assay for extrapulmonary tuberculosis: A meta-analysis.

BackgroundLoop-mediated isothermal amplification (LAMP) is used to detect pulmonary tuberculosis (PTB); however, the diagnostic accuracy of the LAMP assay for extrapulmonary tuberculosis (EPTB) is unclear. We performed a meta-analysis to evaluate the performance of LAMP in the detection of EPTB.MethodsWe searched PubMed, EMBASE, the Cochrane Library, China National Knowledge Infrastructure (CNKI), and the Wanfang database for studies published before Sep 16, 2017. We reviewed studies and compared the performance of LAMP with that of a composite reference standard (CRS) and culture for clinically suspected EPTB. We used a bivariate random-effects model to perform meta-analyses and used meta-regression and subgroup analysis to analyze sources of heterogeneity.ResultsFourteen articles including 24 independent studies (16 compared LAMP to CRS, 8 to culture) of EPTB were identified. LAMP showed a pooled sensitivity of 77% (95% confidence interval (CI) 68–85), specificity of 99% (95% CI 96–100), and area under SROC curves (AUC) of 0.96 (95% CI 0.94–0.97) against CRS. It showed a pooled sensitivity of 93% (95% CI 88–96), specificity of 77% (95% CI 64–86), and AUC of 0.94 (95% CI 0.92–0.96) against culture. The pooled sensitivity, specificity, and AUC of MPB64 LAMP were 86% (95% CI 86–86), 100% (95% CI 100–100), and 0.97 (95% CI 0.95–0.98), respectively, and those of IS6110 LAMP were 75% (95% CI 64–84), 99% (95% CI 90–100), and 0.91 (95% CI 0.88–0.93), respectively, compared with CRS.ConclusionsThese results suggest good diagnostic efficacy of LAMP in the detection of EPTB. Additionally, the diagnostic efficacy of MPB64 LAMP was superior to that of IS6110 LAMP.

Diagnostic accuracy evaluation of the conventional and molecular tests for Spinal Tuberculosis in a cohort, head-to-head study

Early diagnosis of spinal tuberculosis (TB) is hampered by the flaws of conventional tests. The aim of this study was to assess the value of new and existing molecular tests in a prospective, head-to-head cohort study. Specimens were consecutively collected from spinal TB suspects in four hospitals in Beijing, China. Smear, culture, histopathology, Xpert MTB/RIF (Xpert), and MeltPro TB assays were performed in parallel using the same specimen from each patient. Drug-susceptibility testing (DST) was conducted on the isolates recovered. In total, 438 suspects were recruited; 319 of them were diagnosed with spinal TB according to the composite reference standard (CRS), which was composed of clinical, laboratory, histopathological, and radiological examinations and 18 months of follow-up. Based on conventional testing, 74.29% of patients were classified as confirmed cases, which increased to 90.6% when Xpert outcomes were integrated. Further, 76.60% of probable and 45.71% of possible cases were re-classified as confirmed cases with Xpert. Xpert (85.27%) produced higher sensitivity than histopathology (73.04%), MeltPro TB (57.68%), culture (51.72%) and smear (24.45%) (all P <0.001). Xpert was 100% concordant with phenotypic DST regarding rifampicin resistance detection. The sensitivity and specificity of MeltPro TB for rifampicin resistance detection were 100% and 97.96%, respectively, and 95.00% and 93.88% for isoniazid resistance detection. New molecular tests demonstrated excellent efficiency for spinal TB diagnosis in this cohort study, so their application as initial diagnostic tools would greatly increase the proportion of confirmed cases and dramatically reduce the delay of appropriate treatment. An updated laboratory testing algorithm of the disease is desirable.

Experimental platform utilising melting curve technology for detection of mutations in Mycobacterium tuberculosis isolates

Tuberculosis (TB) remains one of the most deadly infections with approximately a quarter of cases not being identified and/or treated mainly due to a lack of resources. Rapid detection of TB or drug-resistant TB enables timely adequate treatment and is a cornerstone of effective TB management. We evaluated the analytical performance of a single-tube assay for multidrug-resistant TB (MDR-TB) on an experimental platform utilising RT-PCR and melting curve analysis that could potentially be operated as a point-of-care (PoC) test in resource-constrained settings with a high burden of TB. Firstly, we developed and evaluated the prototype MDR-TB assay using specimens extracted from well-characterised TB isolates with a variety of distinct rifampicin and isoniazid resistance conferring mutations and nontuberculous Mycobacteria (NTM) strains. Secondly, we validated the experimental platform using 98 clinical sputum samples from pulmonary TB patients collected in high MDR-TB settings. The sensitivity of the platform for TB detection in clinical specimens was 75% for smear-negative and 92.6% for smear-positive sputum samples. The sensitivity of detection for rifampicin and isoniazid resistance was 88.9 and 96.0% and specificity was 87.5 and 100%, respectively. Observed limitations in sensitivity and specificity could be resolved by adjusting the sample preparation methodology and melting curve recognition algorithm. Overall technology could be considered a promising PoC methodology especially in resource-constrained settings based on its combined accuracy, convenience, simplicity, speed, and cost characteristics.

Improvement in the Diagnosis of Tuberculosis Combining Mycobacterium Tuberculosis Immunodominant Peptides and Serum Host Biomarkers

Pulmonary tuberculosis (PTB) is a public health problem with 10.4 million new cases reported in 2017 (1). According to the World Health Organization (WHO), accurate diagnostic tests based in serum biomarkers to detect new cases of tuberculosis are necessary. To evaluate antibodies against Mycobacterium. tuberculosis (Mtb) peptides (Ab-Mtb) and three soluble host biomarkers by ELISA serial multiple test in sera from non-infected controls (NIC, n=31), latent tuberculosis (LTB, n=37) and PTB (n=28) patients in a diagnosis tuberculosis assay. Levels of four Ab-Mtb peptides derived from Mtb and three host response molecules in serum from NIC, LTB and PTB were evaluated by ELISA as tuberculosis biomarkers. Multiple comparisons tests, determination of diagnostic values and ROC curves were performed. Serial and parallel multiple tests were performed with the biomarkers with the highest discriminatory capacity to improve diagnostic values of the test. We found significant differences between biomarkers levels in PTB comparing LTB and NIC to all candidate biomarkers; peptides P12033, P12037, and serum biomarkers such as sCD14 and chemokine CXCL9 showed the best sensitivity and specificity, the highest discriminatory power, and the best area under the curve (AUC) individually. In serial multiple tests, P12037 and sCD14 together have 92% of sensitivity and 91% of specificity, with positive and negative likelihood ratios greater than10. Ab-Mtb peptide P12037 and sCD14 could be applied in a diagnostic test for suspected PTB to improve accuracy and time to diagnosis and could be implemented in a POCT device which can be affordable.

Evaluation of risk factors for false-negative results with an antigen-specific peripheral blood-based quantitative T cell assay (T-SPOT®. TB) in the diagnosis of active tuberculosis: A large-scale retrospective study in China.

ObjectiveTo investigate the diagnostic efficacy of an interferon-γ release assay, T-SPOT®.TB, for diagnosing active tuberculosis (TB) and to identify risk factors for false-negative results.MethodsThis retrospective study enrolled consecutive patients with active TB and with non-TB respiratory diseases to evaluate the risk factors for false-negative results when using the T-SPOT®.TB assay for the diagnosis of active TB. Patients with active TB were categorized as having confirmed pulmonary TB, clinically diagnosed pulmonary TB or extrapulmonary TB (EPTB).ResultsThis study analysed 4964 consecutive patients; 2425 with active TB and 2539 with non-TB respiratory diseases. Multivariate logistic regression analyses identified the following five factors that were all associated with an increased false-negative rate with the T-SPOT®.TB assay: increased age (odds ratio [OR] 1.018; 95% confidence interval [CI] 1.013, 1.024); decreased CD8+ count (OR 0.307; 95% CI 0.117, 0.803); negative sputum acid-fast bacilli (AFB) smear staining (OR 1.821; 95% CI 1.338, 2.477); negative mycobacterial cultures (OR 1.379; 95% CI 1.043, 1.824); and absence of EPTB (OR 1.291; 95% CI 1.026, 1.623).ConclusionsIncreased age, decreased CD8+ count, negative sputum AFB smear results, negative sputum mycobacterial cultures and absence of EPTB might lead to an increased false-negative rate when using the T-SPOT®.TB assay.

Interferon-Gamma Release Assay is Not Appropriate for the Diagnosis of Active Tuberculosis in High-Burden Tuberculosis Settings: A Retrospective Multicenter Investigation.

Background:Interferon-gamma release assay (IGRA) has been used in latent tuberculosis (TB) infection and TB diagnosis, but the results from different high TB-endemic countries are different. The aim of this study was to investigate the value of IGRA in the diagnosis of active pulmonary TB (PTB) in China.Methods:We conducted a large-scale retrospective multicenter investigation to further evaluate the role of IGRA in the diagnosis of active PTB in high TB-epidemic populations and the factors affecting the performance of the assay. All patients who underwent valid T-SPOT.TB assays from December 2012 to November 2015 in six large-scale specialized TB hospitals in China and met the study criteria were retrospectively evaluated. Patients were divided into three groups: Group 1, sputum culture-positive PTB patients, confirmed by positive Mycobacterium tuberculosis sputum culture; Group 2, sputum culture-negative PTB patients; and Group 3, non-TB respiratory diseases. The medical records of all patients were collected. Chi-square tests and Fisher's exact test were used to compare categorical data. Multivariable logistic analyses were performed to evaluate the relationship between the results of T-SPOT in TB patients and other factors.Results:A total of 3082 patients for whom complete information was available were included in the investigation, including 905 sputum culture-positive PTB cases, 914 sputum culture-negative PTB cases, and 1263 non-TB respiratory disease cases. The positive rate of T-SPOT.TB was 93.3% in the culture-positive PTB group and 86.1% in the culture-negative PTB group. In the non-PTB group, the positive rate of T-SPOT.TB was 43.6%. The positive rate of T-SPOT.TB in the culture-positive PTB group was significantly higher than that in the culture-negative PTB group (χ2 = 25.118, P < 0.01), which in turn was significantly higher than that in the non-TB group (χ2 = 566.116, P < 0.01). The overall results were as follows: sensitivity, 89.7%; specificity, 56.37%; positive predictive value, 74.75%; negative predictive value, 79.11%; and accuracy, 76.02%.Conclusions:High false-positive rates of T-SPOT.TB assays in the non-TB group limit the usefulness as a single test to diagnose active TB in China. We highly recommend that IGRAs not be used for the diagnosis of active TB in high-burden TB settings.

Young Investigator: A Rare Case of Crohnʼs Disease-Associated Aortitis

RESULTS: A 33 year-old woman with history of Crohn's disease complicated by enteropathic arthropathies presented with symptoms of complete gastrointestinal obstruction. She had history of ongoing ileocolitis despite therapy with certolizumab. Due to persistent obstruction symptoms, she underwent an uneventful laparoscopic ileocecectomy. On post-operative day number two, she developed fever, chest pain, tachycardia and shortness of breath. Her WBC was elevated to 10 K/microL, with CRP 10 mg/L, total IgG and IgG2 noted to be elevated with normal IgG4 levels. Interferon release assay for tuberculosis was negative. A chest CT revealed ascending aortitis, which was later confirmed with MR angiography. She was started on high-dose corticosteroids and ultimately transitioned to infliximab (5 mg/kg) and subcutaneous methotrexate with resolution of aortitis on follow-up imaging. While her aortitis has remained in remission, her intestinal Crohn's symptoms and arthralgias are recurring despite recently increasing infliximab dose to 10mg/kg every 6 weeks, with therapeutic levels and no evidence of anti-infliximab antibodies. CONCLUSION(S): Extraintestinal manifestations (EIM) of inflammatory bowel disease are commonly found in approximately one-third of patients. They typically manifest as peripheral or axial arthropathies, dermatologic manifestations including pyoderma gangrenosum and erythema nodosum, ocular involvement including uveitis or episcleritis, and primary sclerosing cholangiopathy. While very uncommon, there also appears to be some degree of overlap between Crohn's disease and large vessel vasculitides. Initial evaluation should exclude diagnoses of Takayasu's arteritis, Behcet's disease, sarcoidosis, ankylosing spondylitis, and connective tissue disorders. Infectious etiologies such as tuberculosis and syphilis should also be eliminated. Upon exclusion of these disease processes causing aortic inflammation, the diagnosis of a Crohn's-related EIM should be considered. Only a few cases of aortitis associated with Crohn's disease have been reported in the literature but this appears to be a very rare phenomenon. Severe cases of aortitis carry the risk of aortic rupture when inflammation is left untreated. Fortunately, aortitis appears to be readily responsive to high dose corticosteroid therapy, as was the case in our patient. If significant valvular disease is present (i.e. severe aortic regurgitation), this may require valvular replacement surgery. Interestingly, there have been reports of Crohn's-related aortitis developing in the setting of anti-TNF therapy, again requiring corticosteroid therapy for resolution. As such, we will need to monitor our patient closely to detect any signs of recurrent aortitis.

Use of Xpert MTB/RIF Assay in Rural Health Facilities in Southern Ethiopia

Background: Tuberculosis (TB) is the leading cause of morbidity and mortality in Ethiopia. We describe the performance of the Xpert-MTB/RIF assay for presumptive TB in health facilities that refer samples to a regional reference laboratory. Methods: A cross-sectional study was conducted in patients with presumptive TB from 1st April 2015 to 30th August 2016. The study was performed in Gambo Hospital, Ethiopia. The samples sent to the referral laboratory were analyzed according to national protocols on the diagnosis of TB and multidrug resistant-TB in children with presumptive TB. Results: We studied one sample each from 309 unique patients; 197 (63.8%) were less than 14 years old, and 165 (53.4%) were male. The most commonly analyzed sample was gastric aspiration (n=144, 46.6%) followed by sputum (n=92, 29.8%). Gastric aspiration was performed mainly in children (98.6%, 142/144; p<0.001), while peritoneal effusion (94.4%, 17/18; p<0.001), pleural effusion (80.8%, 21/26; p<0.001), lymph node (63.6%, 14/22; p=0.01), and sputum (56/92, 60.9%; p<0.001) were performed mainly in adults. For 10 samples, the results were not available from the referral laboratory. The samples were positive for TB by Xpert MTB/RIF in 22.4% (67/299) of the samples. Only one sample was resistant to rifampicin (0.3%). The results of Xpert MTB/RIF were positive in 76.2% (16/21) of the lymph node samples (p<0.001), 22.3% of the gastric aspiration samples, 20.0% (31/139) of the sputum samples, 1.5% (1/17) of the ascites fluid samples, and 0.0% (0/25) of the pleural effusions (p=0.002). Conclusion: Xpert MTB/RIF facilitates the diagnosis of TB, with microbiological confirmation, in up to 20% of the samples.

Efficacy and role of Xpert® Mycobacterium tuberculosis/rifampicin assay in urinary tuberculosis.

Introduction:The aim was to study the accuracy of Xpert® (Cepheid Inc., Sunnyvale, CA, USA) Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay as compared to a composite gold standard (urine culture, imaging, and biopsy) and to asses its utility as the initial test compared to smear microscopy to diagnose urinary tuberculosis.Methods:This prospective study included adult patients suspected to have urinary tuberculosis from March 2014 to December 2017. Three urine samples were collected from each patient and were subjected to Xpert MTB/RIF assay, acid-fast bacillus (AFB) smear microscopy, and liquid media (BACTEC Mycobacteria Growth Indicator Tube [MGIT] 960) culture. Imaging and tissue biopsies were performed as clinically indicated. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using the bootstrap method for 95% confidence intervals for the Xpert assay.Results:Xpert MTB/RIF assay was found to be superior to the currently best available light-emitting diode fluorescent smear microscopy as the initial test for urinary tuberculosis (sensitivity of 69.09% vs. 32.72%). The Xpert MTB/RIF polymerase chain reaction test was found to have a moderate sensitivity (69.09%) and high specificity (100%) as compared to the composite reference standard. The sensitivity of liquid AFB culture MGIT 960 as compared to the reference standard was 90.32%.Conclusions:Xpert MTB/RIF assay on an early morning first void urine specimen can replace smear microscopy as the initial diagnostic test for urinary tuberculosis.

A novel inexpensive electrochemical sensor for pyrazinoic acid as a potential tool for the identification of pyrazinamide-resistant Mycobacterium tuberculosis.

Tuberculosis (TB) is a significant cause of morbidity and mortality worldwide. The patient compliance with the long treatment regimens is essential for successful eradication. Pyrazinamide (PZA) shortens these regimens from 9 to 6 months, and therefore, improves treatment completion rates. Although PZA is a first-line medication for the treatment of TB, no simple or reliable assay to determine PZA resistance is yet available. In the presence of PZA, only susceptible Mycobacterium tuberculosis strains release pyrazinoic acid (POA). Therefore, the measurement and quantification of released POA is an indicator of PZA resistance. Two electrochemical sensors were constructed and tested with alternative working electrodes in conjunction with a portable potentiostat to measure the current produced when a potential difference of 2 V is applied to varying concentrations of POA in controlled solutions. The large (13.2 mm) electrochemical sensor was able to detect POA at a minimum concentration of 40 μM to a statistically significant level (P = 0.0190). Similar graphical trends were obtained when testing the electrochemical sensor in the supernatant of a negative microscopic observation drug susceptibility assay culture, irrespective of the presence of PZA. Inexpensive and reusable electrochemical sensors with a portable potentiostat are a promising tool for the detection of POA, a biomarker of PZA susceptible M. Tuberculosis.

Comparison of Interferon-γ Release Assays and the Tuberculin Skin Test for Diagnosis of Tuberculosis in Human Immunodeficiency Virus: A Systematic Review.

BackgroundIt remains uncertain if interferon-γ release assays (IGRAs) are superior to the tuberculin skin test (TST) for the diagnosis of active tuberculosis (TB) or latent tuberculosis infection (LTBI) in immunosuppressed populations including people with human immunodeficiency virus (HIV) infection. The purpose of this study was to systematically review the performance of IGRAs and the TST in people with HIV with active TB or LTBI in low and high prevalence TB countries.MethodsWe searched the MEDLINE database from 1966 through to January 2017 for studies that compared results of the TST with either the commercial QuantiFERON-TB Gold in Tube (QFTGT) assay or previous assay versions, the T-SPOT.TB assay or in-house IGRAs. Data were summarized by TB prevalence. Tests for concordance and differences in proportions were undertaken as appropriate. The variation in study methodology was appraised.ResultsThirty-two studies including 4,856 HIV subjects met the search criteria. Fourteen studies compared the tests in subjects with LTBI in low TB prevalence settings. The QFTGT had a similar rate of reactivity to the TST, although the first-generation version of that assay was reactive more commonly. IGRAs were more frequently positive than the TST in HIV infected subjects with active TB. There was considerable study methodology and population heterogeneity, and generally low concordance between tests. Both the TST and IGRAs were affected by CD4 T-cell immunodeficiency.ConclusionOur review of comparative data does not provide robust evidence to support the assertion that the IGRAs are superior to the TST when used in HIV infected subjects to diagnose either active TB or LTBI.

Cytokine profiling in healthy children shows association of age with cytokine concentrations

Cytokine-based diagnostic assays are increasingly used in research and clinical practice. Assays developed for adults such as the interferon-gamma release assay for tuberculosis show inferior performance in children. Limited evidence suggests that release of cytokines is influenced by age but normal ranges of cytokines in children are lacking. Whole blood of healthy children (0–12 years) undergoing elective/diagnostic procedures was stimulated with SEB, PHA, Candida albicans for 24 hours or left unstimulated. Concentrations of eight cytokines were measured by multiplex bead-based immunoassays and associations with age and other factors quantified by regression analysis. 271 children (median age 5.2 years) were included. In unstimulated samples IL-1ra, IP-10 and TNF-α concentrations decreased by up to −60% with age. Following antigen stimulation, an age-associated increase (ranging from +90% to +500%) was observed for all cytokines except IL-1ra (significant for IL-4, IFN-γ and TNF-α). Inter-individual variability in cytokine concentrations was large with a coefficient of variation ranging from 42% to 1412%. Despite inter-individual variation age was identified as a strong influencing factor of cytokine concentrations. Age-specific normal values need to be considered for cytokine-based diagnostic purposes. These results are relevant for development of novel cytokine-based diagnostic assays and for optimal dosing of therapeutic agents targeting cytokines.

Development and evaluation of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis

BackgroundRed deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. This study aims to develop an in-house sandwich ELISA for deer IFNγ, based on the cross-reactivity of the antibodies to both cervid and bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected red deer in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The positive control stimulant used in this study was pokeweed mitogen, which resulted in a consistent induction of IFNγ in samples from red deer, thus allowing the interpretation of the assay.ResultsThe percentage of animals correctly classified by this technique as M. bovis non-infected was 100%. The detection of infected animals as positive was high and ranged widely depending upon the antigen and the cut-off value applied, as well as the time after infection. Our findings indicate that this protocol may serve as a reliable assay for the antemortem diagnosis of TB from the initial stage of M. bovis-infection, and may also be adequately sensitive.ConclusionsThe suggested optimal antigens and cut-off are bPPD, p22 and the combination of ESAT-6/CFP-10 and Rv3020c with a 0.05 Δ optical density, which yielded a up to 100% correct classification of TB positive and negatve red deer under our experimental conditions. This technique will aid in TB testing of farmed and translocated deer. Future studies should evaluate the ability of this IFNγ assay to detect specific responses under field conditions.

A Rare Presentation of Eosinophilic Granulomatosis with Polyangiitis with Diffuse Lymphadenopathy

A Rare Presentation of Eosinophilic Granulomatosis with Polyangiitis with Diffuse Lymphadenopathy

Diagnosis of tuberculous pleurisy with combination of adenosine deaminase and interferon-γ immunospot assay in a tuberculosis-endemic population: A prospective cohort study.

The aim of this study was to identify the optimal cut-off value of T cell enzyme-linked immunospot assay for tuberculosis (T-SPOT.TB) and evaluate its diagnostic performance alone (in the peripheral blood) or in combination with the adenosine deaminase (ADA) activity test (in peripheral blood and the pleural fluid) in patients with tuberculous pleurisy.Adult patients presenting with pleural effusion were included in this prospective cohort study. Tuberculous pleurisy was diagnosed by T-SPOT.TB in peripheral blood and a combination of T-SPOT.TB and ADA activity test in pleural fluid and peripheral blood. Receiver operating characteristic (ROC) curve in combination with multivariate logistic regression was used to evaluate the diagnostic performance of the assays.Among a total of 189 patients with suspected tuberculous pleurisy who were prospectively enrolled in this study, 177 patients were validated for inclusion in the final analysis. ROC analysis revealed that the area under the ROC curve (AUC) for T-SPOT.TB in pleural fluid and peripheral blood was 0.918 and 0.881, respectively, and for the ADA activity test in pleural fluid was 0.944. In addition, 95.5 spot-forming cells (SFCs)/2.5 × 105 cells were determined as the optimal cut-off value for T-SPOT.TB in pleural fluid. Parallel combination of T-SPOT.TB and ADA activity test in pleural fluid showed increased sensitivity (96.9%) and specificity (87.5%), whereas serial combination showed increased specificity (97.5%). The combination of 3 assays had the highest sensitivity at 97.9%, with an AUC value of 0.964.T-SPOT.TB in pleural fluid performed better than that in peripheral blood and the ADA activity test in pleural fluid for tuberculous pleurisy diagnosis. The optimal cut-off value of T-SPOT.TB in pleural fluid was 95.5 SFCs/2.5 × 105 cells. Combination of 3 assays might be a promising approach for tuberculous pleurisy diagnosis.