Diagnosis Of Mycobacterium Tuberculosis Infection Research Articles

Case report: Difficult diagnosis of Mycobacterium tuberculosis infection in patients after allogeneic hematopoietic stem cell transplantation: two case reports and a literature review

BackgroundMycobacterium tuberculosis (MTB) is a relatively infrequent infection encountered during hematopoietic stem-cell transplantation (HSCT). The identification of MTB following HSCT remains a complex task, with delayed detection and misdiagnosis potentially resulting in unfavorable outcomes. Metagenomic next-generation sequencing (mNGS) represents a novel, highly sensitive, and rapid diagnostic tool in clinical settings for discerning intricate infections and detecting exceedingly rare pathogensMethodsWith the aid of mNGS, we diagnosed MTB in the lymph nodes and lungs of two patients with hematological diseases following allogeneic peripheral blood hematopoietic stem cell transplantation. Both patients presented with a fever, localized symptoms, and clinical signs. Following inconclusive results from routine tests, impractical biopsy procedures, and unsuccessful responses to empirical treatments, mNGS was employed as a final recourse, revealing DNA fragments of MTB in blood samples.ResultsThe diagnoses were ultimately confirmed in conjunction with additional clinical evidence. The application of mNGS in MTB cases after allogeneic HSCT has rarely been reported. The mNGS technique can provide a prompt and highly sensitive indication leading to the definitive diagnosis of MTB in complex post-transplant scenarios.

EBUS-GS with the GeneXpert MTB/RIF assay for diagnosis of Mycobacterium tuberculosis infection of isolated pulmonary nodules

ObjectiveInvestigate the use of endobronchial ultrasonography with a guide sheath (EBUS-GS) combined with Gene Xpert MTB/RIF (Xpert) for diagnosis of Mycobacterium tuberculosis (MTB) infection in isolated pulmonary nodules.MethodsPatients who had isolated pulmonary nodules and unknown diagnoses at our institution from October 2020 to December 2021 were prospectively examined using EBUS-GS and Xpert. The diagnostic values of using EBUS-GS or bronchoalveolar lavage fluid (BALF) with acid-fast staining, MGIT 960 culture, pathological examination, and Xpert for isolated pulmonary nodules caused by MTB infection were compared using receiver operating characteristic (ROC) analysis.ResultsThere were 135 patients, 64 with isolated pulmonary tuberculomas and 71 with non-tuberculous lesions. The sensitivity of EBUS-GS with Xpert was significantly higher than BALF with Xpert (57.81% vs. 34.78%, P = 0.017). Use of EBUS-GS with Xpert and MGIT 960 culture further increased the sensitivity to 62.50% (95%CI 50.64–74.36) and increased the specificity to 100%. The AUC values of BALF with MGIT 960 culture was 0.663(95%CI 0.543–0.783) and BALF with Xpert was 0.674 (95%CI 0.556–0.792). The AUC values of EBUS-GS with MGIT 960 culture was 0.680 (95%CI 0.554–0.743), with pathological examination was 0.713 (95%CI 0.573–0.760), and with Xpert was 0.789 (95%CI 0.655–0.829).ConclusionUse of EBUS-GS with Xpert had high sensitivity and specificity in the diagnosis of isolated pulmonary tuberculoma. This method has significant potential for use in clinical practice.

Inflated Gamma Interferon Response with QuantiFERON-TB Gold Plus Using the Automated Liaison XL Analyzer: a Testing Algorithm To Mitigate False-Positive Results in Low-Incidence Settings.

The Liaison XL chemiluminescence immunoassay (CLIA) analyzer allows total automation of gamma interferon (IFN-γ) measurement for the QuantiFERON-TB Gold Plus assay (QFT-Plus) that is used to diagnose Mycobacterium tuberculosis infection. To evaluate CLIA accuracy, plasma samples from 278 patients undergoing QFT-Plus testing were first tested with an enzyme-linked immunosorbent assay (ELISA; 150 negatives and 128 positives) and subsequently with the CLIA. Three strategies to mitigate false-positive CLIA results were investigated in 220 samples with borderline-negative ELISA results (TB1 and/or TB2, 0.1 to 0.34 IU/mL). The Bland-Altman plot of difference versus average of the two IFN-γ measurements in the Nil and antigen (TB1 and TB2) tubes showed higher IFN-γ measurements across the range of values with the CLIA than with the ELISA. Bias was 0.21 IU/mL (standard deviation, 0.61; 95% confidence interval [CI], -1.0 to 1.41). Linear regression of difference versus average had a slope of 0.08 (95% CI, 0.05 to 0.10), which was significantly nonzero (P < 0.0001). The CLIA had positive and negative percent agreement levels with the ELISA of 91.7% (121/132) and 95.2% (139/146), respectively. In borderline-negative samples tested with ELISA, CLIA was positive in 42.7% (94/220). CLIA with a standard curve resulted in 36.4% (80/220) positivity. Retesting CLIA false positives (TB1 or TB2 range, 0 to ≤1.3 IU/mL) with ELISA reduced false positives by 84.3% (59/70). Retesting with CLIA reduced the false-positive rate by 10.4% (8/77). Implementing the Liaison CLIA for QFT-Plus in low-incidence settings risks falsely elevating conversion rates and overburdening clinics and overtreating patients. Confirming borderline positives with ELISA is a viable strategy to mitigate false-positive CLIA results.

Long-term economic evaluation of the recombinant Mycobacterium tuberculosis fusion protein (EC) test for the diagnosis of Mycobacterium tuberculosis infection.

Background: Tuberculosis continues to be a significant global burden. Purified protein derivative of tuberculin (TB-PPD) is one type of tuberculin skin test (TST) and is used commonly for the auxiliary diagnosis of tuberculosis. The recombinant Mycobacterium tuberculosis fusion protein (EC) test is a new test developed in China. Objective: Evaluate the long-term economic implications of using the EC test compared with the TB-PPD test to provide a reference for clinical decision-making. Methods: The target population was people at a high risk persons of being infected with Mycobacterium tuberculosis. The outcome indicator was quality-adjusted life years (QALY). A cost-utility analysis was used to evaluate the long-term economic implications of using the EC test compared with the TB-PPD test. We employed a decision tree-Markov model from the perspective of the whole society within 77years. Results: Compared with the TB-PPD test, the EC test had a lower cost but higher QALY. The incremental cost-utility ratio was -119,800.7381 CNY/QALY. That is, for each additional QALY, the EC test could save 119,800.7381 CNY: the EC test was more economical than the TB-PPD test. Conclusion: Compared with the TB-PPD test, the EC test would be more economical in the long term for the diagnosis of M. tuberculosis infection according our study.

Recombinant mycobacterium tuberculosis fusion protein for diagnosis of mycobacterium tuberculosis infection: a short-term economic evaluation.

Recombinant Mycobacterium tuberculosis fusion protein (EC) was anticipated to be used for the scale-up of clinical application for diagnosis of Mycobacterium tuberculosis infection in China, but it lacked a head-to-head economic evaluation based on the Chinese population. This study aimed to estimate the cost-utility and the cost-effectiveness of both EC and tuberculin pure protein derivative (TB-PPD) for diagnosis of Mycobacterium tuberculosis infection in the short term. From a Chinese societal perspective, both cost-utility analysis and cost-effectiveness analysis were performed to evaluate the economics of EC and TB-PPD for a one-year period based on clinical trials and decision tree model, with quality-adjusted life years (QALYs) as the utility-measured primary outcome and diagnostic performance (including the misdiagnosis rate, the omission diagnostic rate, the number of patients correctly classified, and the number of tuberculosis cases avoided) as the effective-measured secondary outcome. One-way and probabilistic sensitivity analyses were performed to validate the robustness of the base-case analysis, and a scenario analysis was conducted to evaluate the difference in the charging method between EC and TB-PPD. The base-case analysis showed that, compared with TB-PPD, EC was the dominant strategy with an incremental cost-utility ratio (ICUR) of saving 192,043.60 CNY per QALY gained, and with an incremental cost-effectiveness ratio (ICER) of saving 7,263.53 CNY per misdiagnosis rate reduction. In addition, there was no statistical difference in terms of the omission diagnostic rate, the number of patients correctly classified, and the number of tuberculosis cases avoided, and EC was a similar cost-saving strategy with a lower test cost (98.00 CNY) than that of TB-PPD (136.78 CNY). The sensitivity analysis showed the robustness of cost-utility and cost-effectiveness analysis, and the scenario analysis indicated cost-utility in EC and cost-effectiveness in TB-PPD. This economic evaluation from a societal perspective showed that, compared to TB-PPD, EC was likely to be a cost-utility and cost-effective intervention in the short term in China.

Coronin-1A serves as a serum biomarker for supportive diagnosis of Mycobacterium tuberculosis infection.

The severity and spread of tuberculosis, a major burden, can be prevented by more rapid and accurate laboratory diagnosis. The purpose of this study is to systematically explore candidate serum proteins in patients with Mycobacterium tuberculosis infection for further application as novel biomarkers. Our study was performed in two major steps: screening of the literature for potential biomarkers, and then validation of their levels in patients and controls. Many serum/plasma proteins previously reported to be abnormally expressed in patients with tuberculosis between 2012 and 2021 were comprehensively assembled. The biological role in tuberculosis was also predicted for each using the bioinformatics tool STRING. Candidate proteins found to have the same expression in other related diseases were excluded. Subsequently, the serum level of the candidate serum/plasma protein that met the aforementioned criteria was validated by sandwich ELISA; diagnostic performance was analysed by the area under the curve (AUC) of the receiver operating characteristic (ROC). From 103 collected serum/plasma proteins, coronin-1A was found to have abnormal expression only in patients with tuberculosis and was associated with tuberculosis. In addition, the validation of coronin-1A in the serum of patients with pulmonary tuberculosis revealed a higher level than in that of healthy individuals. Furthermore, the area under the ROC curve for diagnostic power of coronin-1A was 0.866, with high sensitivity and specificity at a cut-point of approximately 52.7 ng/mL. We concluded that the level of serum coronin-1A might serve as a novel biomarker for alternative laboratory examination to effectively distinguish patients with tuberculosis from those with other related diseases and healthy individuals.

Portable paper-based electrochemiluminescence test incorporating lateral-flow immunosensors for detection of interferon-γ levels.

Tuberculosis (TB) poses a serious threat to human health and social development. Accurate diagnosis of mycobacterium tuberculosis infection plays a critical role in the prevention and treatment of tuberculosis. Interferon-γ (INF-γ) release assay (IGRA) is currently the only quantitative tuberculosis infection diagnosis method. An accurate, fast, and easily handled INF-γ detection method is the key to obtaining accurate results. Herein, we report a novel paper-based electrochemiluminescence (ECL) method based on lateral flow immunosensors that combines the easy handling characteristics of immunochromatography and the high sensitivity of electrochemiluminescence to detect IFN-γ. To our knowledge this is the first INF-γ detection method that combines immunochromatography with electrochemiluminescence. The paper-based ECL-LFI test consists of a sample pad, conjugation pad (with binding antibody IFN-γ-Ab1 conjugated with ruthenium tripyridine), detection pad (with capture antibody IFN-γ-Ab2 immobilized on nanospheres), absorbent pad, and electrode for signal activation. The ECL signal is obtained by cyclic voltammetry scanning at a speed of 0.1V/s in the detection area of the paper-based ECL-LFI test. In our experiments, the paper-based ECL-LFI test exhibited a minimum detection limit of 2.57pg/mL within 12 min, and a broad detection range of 2.57-5,000pg/mL, with repeatability of 8.10% and stability of 4.97%. With the advantage of high accuracy and sensitivity, easy handling, and low user training requirements, this ECL-LFI test might be used as point-of-care testing (POCT) in the IGRA for tuberculosis diagnosis.

The Interferon-Gamma Release Assay versus the Tuberculin Skin Test in the Diagnosis of Mycobacterium tuberculosis Infection in BCG-Vaccinated Children and Adolescents Exposed or Not Exposed to Contagious TB.

Children have an increased risk of developing active tuberculosis (TB) after exposure to Mycobacterium tuberculosis (M.tb), and they are more likely to develop the most severe forms of TB. Rapid diagnosis and treatment of latent M.tb infection (LTBI) is essential to lessen the devastating consequences of TB in children. The aim of the study was to evaluate TST (tuberculin skin test) and IGRA (interferon-gamma release assay) utility in identifying LTBI in a cohort of Bacille Calmette-Guérin (BCG)-vaccinated Polish children and adolescents exposed or not exposed to contagious TB. In addition, we asked whether quantitative assessment of IGRA results could be valuable in predicting active TB disease. Of the 235 recruited volunteers, 89 (38%) were TST-positive (TST+), 74 (32%) were IGRA-positive (IGRA+), and 62 (26%) were both TST+ and IGRA+. The frequency of TST positivity was significantly higher in the group with (59%) than without TB contact (18%). The percentage of TST+ subjects increased with age from 36% in the youngest children (<2 years) to 47% in the oldest group (>10 years). All positive IGRA results were found solely in the group of children with TB contact. There was a significant increase in the rate of positive IGRA results with age, from 9% in the youngest to 48% in the oldest group. The 10 mm TST cutoff showed good sensitivity and specificity in both TB exposed and nonexposed children and was associated with excellent negative predictive value, especially among nonexposed volunteers. Mean IFN-γ concentrations in IGRA cultures were significantly higher in the group of LTBI compared to the children with active TB disease, both TST+ and TST-. Both TST and IGRA can be used as screening tests for BCG-vaccinated children and adolescents exposed to contagious TB.

How mycobacterium tuberculosis infection could lead to the increasing risks of chronic fatigue syndrome and the potential immunological effects: a population-based retrospective cohort study

BackgroundChronic fatigue syndrome (CFS) has been shown to be associated with infections. Tuberculosis (TB) is a highly prevalent infectious disease. Patients with chronic fatigue syndrome and post-tuberculosis experience similar symptoms. Furthermore, chronic fatigue syndrome and tuberculosis share similar plasma immunosignatures. This study aimed to clarify the risk of chronic fatigue syndrome following the diagnosis of Mycobacterium tuberculosis infection (MTI), by analyzing the National Health Insurance Research Database of Taiwan.Methods7666 patients aged 20 years or older with newly diagnosed Mycobacterium tuberculosis infection during 2000–2011 and 30,663 participants without Mycobacterium tuberculosis infection were identified. Both groups were followed up until the diagnoses of chronic fatigue syndrome were made at the end of 2011.ResultsThe relationship between Mycobacterium tuberculosis infection and the subsequent risk of chronic fatigue syndrome was estimated through Cox proportional hazards regression analysis, with the incidence density rates being 3.04 and 3.69 per 1000 person‐years among the non‐Mycobacterium tuberculosis infection and Mycobacterium tuberculosis infection populations, respectively (adjusted hazard ratio [HR] = 1.23, with 95% confidence interval [CI] 1.03–1.47). In the stratified analysis, the Mycobacterium tuberculosis infection group were consistently associated with a higher risk of chronic fatigue syndrome in the male sex (HR = 1.27, 95% CI 1.02–1.58) and age group of ≥ 65 years old (HR = 2.50, 95% CI 1.86–3.38).ConclusionsThe data from this population‐based retrospective cohort study revealed that Mycobacterium tuberculosis infection is associated with an elevated risk of subsequent chronic fatigue syndrome.

Validity of Ziehl-Neelsen and LED Fluorescence Microscopy Technique in the Diagnosis of Mycobacterium Tuberculosis Infection among HIV Patients in Lafia, Nigeria, 2017

Tuberculosis (TB) is a public health problem caused by the Mycobacterium tuberculosis complex. An estimated 70% of TB patients living with HIV are from sub-Saharan Africa largely depends on direct smear microscopy light microscopy for TB diagnosis, which has low Sensitivity, especially among HIV patients. Hence, we conducted this study to evaluate the Performance of Auramine O LED Fluorescence microscopy and the Ziehl-Neelsen technique in Tuberculosis among HIV-positive patients. The study was conducted using a cross-sectional design among 107 consecutively selected HIV/AIDS patients with presumptive Tuberculosis attending ART clinic in Dalhatu Araf Specialist Hospital (DASH), Lafia, Nasarawa State. Three samples were collected from each patient in two visits. Each sample was examined using ZN, LED FM, and Gene-Xpert. Data on socio-demographic characteristics was collected from the subject participant. Univariate and bivariate analyses were done using MS Excel and Epi Info. The Sensitivity and Specificity were compared using McNemar’s chi-square. Seventy-three (68%) patients were female, 33 (30.8%) were in the age group 28-32 years, and 61 (56%) were employed. Ziehl-Neelsen and LED fluorescent microscopy respectively yielded 11 (10.3%) and 15 (14.0%) positive results, while Gene-Xpert gave 18 (16.3%). The Sensitivity for direct ZN and LED FM were 61.1% and 83.3 %, respectively (P-value 0.01) with 100% specificity for both methods. LED microscopy has a much higher sensitivity than ZN microscopy and would be a better alternative in the diagnosis of Tuberculosis in high HIV settings where the use and expertise with culture and Gene-Xpert are limited. Keywords: HIV, LED Fluorescence, Sensitivity, Specificity, Ziehl-Neelsen.

Structural implications of lipoarabinomannan glycans from global clinical isolates in diagnosis of Mycobacterium tuberculosis infection

In Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure–function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates (HN878, EAI, and IO) and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAM-methylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal β-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has ∼50% of α−arabinofuranose -(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM.

Assessment of delayed tuberculosis diagnosis preceding diagnostic confirmation among tuberculosis patients attending Isiolo County level four hospital, Kenya.

Introductiondelayed diagnosis of Mycobacterium tuberculosis infection leads to accelerated individual to individual transmission. This study evaluated this aspect of delayed diagnosis among patients visiting Isiolo level four hospital in northern Kenya.Methodsthis was a cross-sectional cohort study conducted during January, 2018-January, 2019 with systematically sampled 172 tuberculosis (TB) patients. Epidemiological and clinical characteristics were abstracted from records to serve as independent variables. Outcome variable was delayed diagnosis dichotomised into < 21 or > 21 days and treated as a binary outcome. Pre-tested interviewer-administered questionnaires, focused group discussions, and key informant interview guides were used to collect relevant information.Resultsmost (n=89, 57.8%) of the TB diagnosis fell in the category of > 21 day delay. Overall, among all patients, delay in days constituted a median of 27.6, a mean of 37.3 ± 57 days (range 0-414 days). Factors associated with delayed diagnosis (happening > 21 days) included (i) use of dispensary and private health facilities, (OR=4.3, 95% CI: 1.44,13.14; P=0.009) and (OR= 4.9, 95% CI: 1.64, 14.73; P=0.004), respectively (ii) Self-employed individuals (OR=21.7, 95% CI: 2.47,190.93; P=0.006) and employed individuals (OR=9.9, 95% CI: 1.14, 85.80; P=0.038) (iii) secondary-level education (OR=0.03, 95% CI: 0.01,0.21; P=0.000) and tertiary education (OR=0.033, 95% CI: 0.01, 0.23; P=0.001).Conclusiondelayed diagnosis of TB was found to be associated with health-seeking behaviour of TB patients, proxied by diagnosis facility, occupation, and education levels in our study area. Curtailment of local transmission of M. tuberculosis needs intensified health promotion and education in affected communities complemented with active case findings.

Cost-effectiveness of newer technologies for the diagnosis of Mycobacterium tuberculosis infection in Brazilian people living with HIV

Tuberculosis is the leading cause of death among people living with HIV (PLH). Preventive tuberculosis therapy reduces mortality in PLH, especially in those with a positive tuberculin skin test (TST). New, more specific technologies for detecting latent tuberculosis infection (LTBI) are now commercially available. We sought to analyse the cost-effectiveness of four different strategies for the diagnosis of LTBI in PLH in Brazil, from the Brazilian public health care system perspective. We developed a Markov state-transition model comparing four strategies for the diagnosis of LTBI over 20 years. The strategies consisted of TST with the currently used protein purified derivative (PPD RT 23), two novel skin tests using recombinant allergens (Diaskintest [Generium Pharmaceutical, Moscow, Russia] and EC [Zhifei Longcom Biologic Pharmacy Co., Anhui, China]), and the QuantiFERON-TB-Gold-Plus (Qiagen, Hilden, Germany). The main outcome was cost (in 2020 US dollars) per quality-adjusted life years (QALY). For the base case scenario, the Diaskintest was dominant over all other examined strategies. The cost saving estimate per QALY was US $1375. In sensitivity analyses, the Diaskintest and other newer tests remained cost-saving compared to TST. For PLH, TST could be replaced by more specific tests in Brazil, considering the current national recommendations.

Performance of the Roche cobas MTB Assay for the Molecular Diagnosis of Pulmonary Tuberculosis in a High HIV Burden Setting

Increased access to and improved sensitivities of methods for diagnosing Mycobacterium tuberculosis infection and detecting rifampicin and isoniazid resistance are needed. Herein, the performance of the new cobas MTB assay for use on cobas 6800/8800 Systems (Roche) was assessed and compared with two other commercial assays: RealTime MTB (Abbott), and Xpert MTB/RIF (Cepheid). Molecular PCR-based assays were conducted on sputum specimens from individuals with presumptive and confirmed tuberculosis (n=294) from two clinical facilities in South Africa between December 2016 and October 2017. Liquid mycobacterial culture was the reference. Test sensitivities were 94.7% (95% CI, 88%-98%), 92.6% (95% CI, 85%-97%), and 91.6% (95% CI, 84%-96%) for cobas MTB, RealTime MTB, and Xpert MTB/RIF assays, respectively. cobas MTB sensitivity was unaffected by HIV coinfection (95.7%; 95% CI, 88%-99%; n=176) and sediment testing (94.7%; 95% CI, 88%-98%). Sensitivities were 81.8% (95% CI, 60%-95%), 72.7% (95% CI, 50%-89%), and 72.7% (95% CI, 50%-89%) among smear-negative, culture-positive individuals (n=221) for cobas MTB, RealTime MTB, and Xpert MTB/RIF assays, respectively. cobas MTB specificity was 95.7% (95% CI, 89%-99%) and 99% (95% CI, 94%-100%) among HIV coinfected and uninfected individuals, respectively. The cobas 6800/8800 system is already implemented in South Africa for high-throughput HIV viral load testing, making it suitable for integrated HIV/tuberculosis diagnostics.

Accuracy of QuantiFERON-TB Gold Plus Test for Diagnosis of Mycobacterium tuberculosis Infection in Children.

Compared to its predecessor QuantiFERON-TB Gold In Tube (QFT-IT), QuantiFERON-TB Gold Plus (QFT-Plus) contains an additional antigen tube (TB2), stimulating both CD4+ and CD8+ T cells. The ability to discriminate CD4+ and CD8+ responses is suggested to be useful in differentiating stages of Mycobacterium tuberculosis infection. While QFT-Plus has already been evaluated in adults, there are not enough data in children evaluated for suspected active tuberculosis (TB) or latent TB infection (LTBI). A prospective cross-sectional study was conducted among children aged 0 to 17 years who were evaluated for suspected active TB or screened for LTBI. All children underwent QFT-Plus and further clinical, radiological, and/or microbiological analyses according to clinical scenario. Of the 198 children enrolled, 43 (21.7%) were tested because of suspicion of active TB. A total of 12/43 (27.9%) were diagnosed with active TB, and among these, 10/12 (83.3%) had a positive QFT-Plus assay. Of the 155 children screened for LTBI, 18 (11.6%) had a positive QFT-Plus, and 5 (2.5%) had an indeterminate result. TB1 and TB2 quantitative responses were not able to discriminate active disease from latent infection. The percent agreement between TB1 and TB2 was 100%. QFT-Plus assay showed good sensitivity for active TB and was particularly useful for the evaluation of children with suspected LTBI, giving a low rate of indeterminate results in this group. More studies are needed to properly evaluate QFT-Plus ability in discriminating active disease from latent infection.

Antibody-Secreting Cells To Diagnose Mycobacterium tuberculosis Infection in Children in Pakistan

Reliance on microbiologic methods to diagnose Mycobacterium tuberculosis infection is a suboptimal approach for children due in part to the paucibacillary nature of the disease. A blood-based biomarker assay, such as the mycobacterial-antibody-secreting cell (MASC) assay, could be a major advance for the field of study of pediatric tuberculosis (TB). Children <15 years of age with clinical concern for TB and age-matched children with no concern for TB were enrolled from outpatient clinics in Karachi, Pakistan. MASC, ferritin, and C-reactive protein (CRP) assays were performed, and results were compared among cases and controls, as well as among children with a case definition of "confirmed TB," "probable TB," or "possible TB." MASC responses were significantly higher among children with TB than among controls (0.41 optical density [OD] versus 0.28 OD, respectively, P < 0.001), and the differences were largely driven by the data from children with confirmed TB (P = 0.002). Ferritin and CRP values were significantly higher among those with confirmed TB than among those with the other disease states and controls (P = 0.004 and P = 0.019, respectively). The use of the MASC assay as a blood-based biomarker for TB disease shows some promise among children with microbiologically confirmed disease; however, the performance characteristics for the majority of young children with unconfirmed TB were suboptimal in this cohort.IMPORTANCE Tuberculosis (TB) in children represents a missed opportunity for diagnosis and preventive therapy. The magnitude or burden of disease in children is not fully understood due to our limitations with respect to exploring sensitive diagnostic algorithms. In a setting of TB endemicity in Pakistan, we carried out a proof-of-concept study to evaluate for the first time the performance of B cell analyses by the use of well-defined diagnostic criteria and NIH consensus guidelines as "culture-confirmed," "probable," and "possible" TB groups. In contrast to detection of serum antibody, we focused on mycobacterial-antibody-secreting cell (MASC) detection as a marker of active disease in children with a strong suspicion of TB. Further work exploring a larger panel of inflammatory biomarkers and enrichment of B cells with the objective of increasing the sensitivity of the current MASC assay would lead to the development of a field-friendly assay for timely diagnosis of childhood TB.

LIOFeron®TB/LTBI: A novel and reliable test for LTBI and tuberculosis

ObjectivesHigh accuracy diagnostic screening tests for tuberculosis (TB) are required to improve the diagnosis of both active TB and latent Mycobacterium tuberculosis (MTB) infection (LTBI). The novel IGRA LIOFeron®TB/LTBI assay was tested and its accuracy was compared to the QuantiFERON®-TB Gold Plus assay. MethodsA total of 389 subjects were enrolled in two cohorts and classified as healthy, active TB or LTBI persons. The blood of all the patients was tested with LIOFeron®TB/LTBI assay, containing MTB alanine dehydrogenase, able to differentiate active TB from LTBI diagnosis. The results obtained with both IGRAs, performed on the same 250 samples, were finally compared. ResultsThe two assays demonstrated an excellent concordance of their results with patients’ diagnosis of MTB infection. ROC analysis for QuantiFERON®-TB Gold Plus showed sensitivity and specificity respectively of 98% and 97% in diagnosing active TB patients and 85% and 94% in diagnosing LTBI subjects. LIOFeron®TB/LTBI assay showed sensitivity and specificity respectively of 90% and 98% in diagnosing active TB patients and 94% and 97% in diagnosing LTBI subjects. ConclusionsThe two IGRAs displayed the same high accuracy in diagnosing MTB infection/TB disease, and LIOFeron®TB/LTBI assay demonstrated higher sensitivity than QuantiFERON®-TB Gold Plus test in LTBI detection.

Contribution of QuantiFERON-TB Gold-in-Tube to the Diagnosis of Mycobacterium tuberculosis Infection in Young Children in a Low TB Prevalence Country.

Introduction: Interferon Gamma Release Assay (IGRA) has proven to be a useful test to evaluate the immune response to Mycobacterium tuberculosis antigens in children over the age of 5 years as an alternative to tuberculin skin testing (TST). Much less is known about its performance in younger children, who are at higher risk for developing tuberculosis (TB) disease after exposure. We aimed to evaluate the accuracy of using IGRA in TB screening in this population.Methods: Children below the age of 5 years at high risk for TB infection were prospectively enrolled, to compare the performance of TST and the QuantiFERON-TB Gold-In-Tube test (QFT). Children were treated in accordance with the diagnosis made at baseline and followed-up for 12 months.Results: We included a total of 60 children of which 97 blood samples were available for analysis. There was 90.72% agreement between TST and QFT (Kappa test 0.59, moderate agreement). With TST as a reference, the QFT positive predictive value was 0.72 and the negative predictive value 0.93. Discordant results were observed with 6% TST+/QFT– paired tests. When we restricted the comparison of TST and QFT to non-BCG-vaccinated children, the degree of agreement was more substantial (95%, Kappa test 0.75) and the negative predictive value was 0.99. We observed 3% discordant TST–/QFT+ results. All children with active TB disease had concordant positive QFT results, with QFT values above 4.00 IU/ml.Conclusion: In a low TB prevalence country, serial testing of QFT was found to produce a moderate agreement with TST results. False positive QFT results would have been eliminated by using a higher cutoff without misdiagnosing the children with TB disease. Some of the false negative QFT results could be explained by false positive TST results on consecutive testing. For now the most prudent approach would be to consider discordant QFT–/TST+ results as false negative QFT results, taking into account the young age of our population and the potential risk for evolution to active TB disease.

Efficacy of ELISA test in diagnosis of Mycobacterium tuberculosis infection

Efficacy of ELISA test in diagnosis of Mycobacterium tuberculosis infection

Boosting effect of IL-7 in interferon gamma release assays to diagnose Mycobacterium tuberculosis infection.

BackgroundA quarter of the world’s population is estimated to be infected with Myobacterium tuberculosis (Mtb). Infection is detected by immune response to M. tuberculosis antigens using either tuberculin skin test (TST) and interferon gamma release (IGRA’s), tests which have low sensitivity in immunocompromised. IL-7 is an important cytokine for T-cell function with potential to augment cytokine release in in-vitro assays. This study aimed to determine whether the addition of IL-7 in interferon-gamma release assays (IGRAs) improves its diagnostic performance of Mtb infection.Methods44 cases with confirmed TB and 45 household contacts without TB were recruited and 1ml of blood was stimulated in two separate IGRA’s tube set: one set of standard Quantiferon TB gold tubes mitogen, TB antigen and TB Nil; one set of customized Quantiferon TB gold tubes with added IL-7. Following IFN-γ and IP-10 release was determined using ELISA.ResultsWe found that the addition of IL-7 led to significantly higher release of IFN-γ in individuals with active TB from 4.2IU/ml (IQR 1.4–6.9IU/ml) to 5.1IU/ml (IQR 1.5–8.1IU/ml, p = 0.0057), and we found an indication of a lower release of both IFN-γ and IP-10 in participants with negative tests.ConclusionsIn TB cases addition of IL-7 in IGRA tubes augments IFN-γ but not IP-10 release, and seems to lower the response in controls. Whether IL-7 boosted IGRA holds potential over standard IGRA needs to be confirmed in larger studies in high and low TB incidence countries.