Xpert MTB/RIF Ultra assay for tuberculosis disease and rifampicin resistance in children

Background Every year, an estimated one million children and young adolescents become ill with tuberculosis, and around 226,000 of those children die. Xpert MTB/RIF Ultra (Xpert Ultra) is a molecular World Health Organization (WHO)‐recommended rapid diagnostic test that simultaneously detects Mycobacterium tuberculosis complex and rifampicin resistance. We previously published a Cochrane Review 'Xpert MTB/RIF and Xpert MTB/RIF Ultra assays for tuberculosis disease and rifampicin resistance in children'. The current review updates evidence on the diagnostic accuracy of Xpert Ultra in children presumed to have tuberculosis disease. Parts of this review update informed the 2022 WHO updated guidance on management of tuberculosis in children and adolescents. Objectives To assess the diagnostic accuracy of Xpert Ultra for detecting: pulmonary tuberculosis, tuberculous meningitis, lymph node tuberculosis, and rifampicin resistance, in children with presumed tuberculosis. Secondary objectives To investigate potential sources of heterogeneity in accuracy estimates. For detection of tuberculosis, we considered age, comorbidity (HIV, severe pneumonia, and severe malnutrition), and specimen type as potential sources. To summarize the frequency of Xpert Ultra trace results. Search methods We searched the Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, three other databases, and three trial registers without language restrictions to 9 March 2021. Selection criteria Cross‐sectional and cohort studies and randomized trials that evaluated Xpert Ultra in HIV‐positive and HIV‐negative children under 15 years of age. We included ongoing studies that helped us address the review objectives. We included studies evaluating sputum, gastric, stool, or nasopharyngeal specimens (pulmonary tuberculosis), cerebrospinal fluid (tuberculous meningitis), and fine needle aspirate or surgical biopsy tissue (lymph node tuberculosis). For detecting tuberculosis, reference standards were microbiological (culture) or composite reference standard; for stool, we also included Xpert Ultra performed on a routine respiratory specimen. For detecting rifampicin resistance, reference standards were drug susceptibility testing or MTBDRplus. Data collection and analysis Two review authors independently extracted data and, using QUADAS‐2, assessed methodological quality judging risk of bias separately for each target condition and reference standard. For each target condition, we used the bivariate model to estimate summary sensitivity and specificity with 95% confidence intervals (CIs). We stratified all analyses by type of reference standard. We summarized the frequency of Xpert Ultra trace results; trace represents detection of a very low quantity of Mycobacterium tuberculosis DNA. We assessed certainty of evidence using GRADE. Main results We identified 14 studies (11 new studies since the previous review). For detection of pulmonary tuberculosis, 335 data sets (25,937 participants) were available for analysis. We did not identify any studies that evaluated Xpert Ultra accuracy for tuberculous meningitis or lymph node tuberculosis. Three studies evaluated Xpert Ultra for detection of rifampicin resistance. Ten studies (71%) took place in countries with a high tuberculosis burden based on WHO classification. Overall, risk of bias was low. Detection of pulmonary tuberculosis Sputum, 5 studies Xpert Ultra summary sensitivity verified by culture was 75.3% (95% CI 64.3 to 83.8; 127 participants; high‐certainty evidence), and specificity was 97.1% (95% CI 94.7 to 98.5; 1054 participants; high‐certainty evidence). Gastric aspirate, 7 studies Xpert Ultra summary sensitivity verified by culture was 70.4% (95% CI 53.9 to 82.9; 120 participants; moderate‐certainty evidence), and specificity was 94.1% (95% CI 84.8 to 97.8; 870 participants; moderate‐certainty evidence). Stool, 6 studies Xpert Ultra summary sensitivity verified by culture was 56.1% (95% CI 39.1 to 71.7; 200 participants; moderate‐certainty evidence), and specificity was 98.0% (95% CI 93.3 to 99.4; 1232 participants; high certainty‐evidence). Nasopharyngeal aspirate, 4 studies Xpert Ultra summary sensitivity verified by culture was 43.7% (95% CI 26.7 to 62.2; 46 participants; very low‐certainty evidence), and specificity was 97.5% (95% CI 93.6 to 99.0; 489 participants; high‐certainty evidence). Xpert Ultra sensitivity was lower against a composite than a culture reference standard for all specimen types other than nasopharyngeal aspirate, while specificity was similar against both reference standards. Interpretation of results In theory, for a population of 1000 children: • where 100 have pulmonary tuberculosis in sputum (by culture): ‐ 101 would be Xpert Ultra‐positive, and of these, 26 (26%) would not have pulmonary tuberculosis (false positive); and ‐ 899 would be Xpert Ultra‐negative, and of these, 25 (3%) would have tuberculosis (false negative). • where 100 have pulmonary tuberculosis in gastric aspirate (by culture): ‐ 123 would be Xpert Ultra‐positive, and of these, 53 (43%) would not have pulmonary tuberculosis (false positive); and ‐ 877 would be Xpert Ultra‐negative, and of these, 30 (3%) would have tuberculosis (false negative). • where 100 have pulmonary tuberculosis in stool (by culture): ‐ 74 would be Xpert Ultra‐positive, and of these, 18 (24%) would not have pulmonary tuberculosis (false positive); and ‐ 926 would be Xpert Ultra‐negative, and of these, 44 (5%) would have tuberculosis (false negative). • where 100 have pulmonary tuberculosis in nasopharyngeal aspirate (by culture): ‐ 66 would be Xpert Ultra‐positive, and of these, 22 (33%) would not have pulmonary tuberculosis (false positive); and ‐ 934 would be Xpert Ultra‐negative, and of these, 56 (6%) would have tuberculosis (false negative). Detection of rifampicin resistance Xpert Ultra sensitivity was 100% (3 studies, 3 participants; very low‐certainty evidence), and specificity range was 97% to 100% (3 studies, 128 participants; low‐certainty evidence). Trace results Xpert Ultra trace results, regarded as positive in children by WHO standards, were common. Xpert Ultra specificity remained high in children, despite the frequency of trace results. Authors' conclusions We found Xpert Ultra sensitivity to vary by specimen type, with sputum having the highest sensitivity, followed by gastric aspirate and stool. Nasopharyngeal aspirate had the lowest sensitivity. Xpert Ultra specificity was high against both microbiological and composite reference standards. However, the evidence base is still limited, and findings may be imprecise and vary by study setting. Although we found Xpert Ultra accurate for detection of rifampicin resistance, results were based on a very small number of studies that included only three children with rifampicin resistance. Therefore, findings should be interpreted with caution. Our findings provide support for the use of Xpert Ultra as an initial rapid molecular diagnostic in children being evaluated for tuberculosis

Specificity of a whole blood IGRA in German nursing students

<p>Abstract</p> <p>Background</p> <p>Interferon-gamma release assays (IGRA) are used for tuberculosis (TB) screening in healthcare workers (HCWs). However, data on specificity of IGRA in serial testing of HCWs is sparse. Therefore the specificity and the negative predictive value of the IGRA - QuantiFERON-TB Gold In-Tube (QFT) - in German nursing students was investigated.</p> <p>Methods</p> <p>194 nursing students at the start of their professional career were tested with the QFT. 14 nursing students were excluded from the specificity analysis, due to exposure to mycobacterium tuberculosis. Two of these subjects were QFT- positive. None of them developed disease during the year of follow-up. A study group of 180 students, all with very low risk of prior TB infection, remained in the specificity analysis. Subjects were monitored for at least two years with respect to the development of active TB disease. IGRA was performed at the start of the training and after one year.</p> <p>Results</p> <p>The mean age of the study group (n = 180) was 23 years (range 18-53) with 70.9% female and 99.4% German born. The specificity of QFT was 98.9% (178/180; 95% CI 0.96-0.99); lowering the cut-off from 0.35 IU/ml to 0.1 IU/ml would have decreased specificity only slightly to 97.8% (176/180; 95% CI 0.94-0.99). Of the 154 nursing students available for re-testing, one student who initially scored positive reverted to negative, and one student initially negative converted to positive. None of the monitored group with initially negative QFT results developed TB disease, indicating a high negative predictive value of the IGRA in this population.</p> <p>Conclusions</p> <p>Following our data, QFT can serve as an effective tool in pre-employment TB screenings for HCWs. As its negative results were stable over time, specificity of the QFT in serial testing of HCWs is high. As the risk of acquiring TB infection in the German healthcare system appears to be low, our data supports the recommendation of performing TB screening only in those HCWs with known contact to TB patients or infectious materials.</p

Is IP-10 a Better Biomarker for Active and Latent Tuberculosis in Children than IFNγ?

Background: The blood based interferon-gamma release assays (IGRA) for the diagnosis of tuberculosis do not discriminate between active TB disease and latent TB infection (LTBI). The search for distinguishing biomarkers therefore continues, as the accurate diagnosis of tuberculosis is particularly challenging in children. IFN-c-inducible protein 10 (IP-10/CXCL10) has recently been evaluated as a marker for active TB in adults with promising results. Aim: To investigate this new biomarker for active TB and LTBI in paediatrics. Method: We measured IP-10 levels using ELISA in supernatants of whole blood samples stimulated with TB-specificantigens and negative control antigen. Results: IP-10 is produced in high levels following mycobacterial antigen stimulation in active TB (n = 17) and LTBI (n = 16) compared to controls (n = 16) and to IFN-c. The baseline levels of IP-10 are increased in active TB and in LTBI, but there is no significant difference of stimulated levels of IP-10 between active TB and LTBI. Conclusions: IP-10 is a biomarker for tuberculosis in children. However like IFNc, IP-10 also does not distinguish between active TB and LTBI

Whole Blood Interferon-Gamma Responses to Mycobacterium tuberculosis Antigens in Young Household Contacts of Persons with Tuberculosis in Uganda

Due to immunologic immaturity, IFN-gamma-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-gamma responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-gamma production in response to mycobacterial antigens between children and adults in Uganda.We studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-gamma production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, n = 80), young children (2 <5 years old, n = 216), older children (5 <15 years old, n = 443) and adults (> or =15 years old, n = 528). We evaluated the relationship between IFN-gamma responses and the tuberculin skin test (TST), and between IFN-gamma responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-gamma responses. Young household contacts demonstrated robust IFN-gamma responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-gamma response.Young children in a TB endemic setting can mount robust IFN-gamma responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection

T-cell-based diagnosis of tuberculosis infection in children in Lithuania: a country of high incidence despite a high coverage with bacille Calmette-Guerin vaccination

<p>Abstract</p> <p>Background</p> <p>Lithuania is a country with a high incidence of tuberculosis (TB), despite a high coverage with bacille Calmette-Guerin (BCG) vaccination. Until now the only method used to detect latent TB infection was the tuberculin skin test (TST). However, TST may have a cross reactivity to the BCG vaccine and to environmental mycobacteria. The aim of this study was to conduct assessments of the diagnostic accuracy of the T-cell based test (T SPOT TB) for TB in children who had previously been BCG vaccinated and compare these with the results of the TST.</p> <p>Methods</p> <p>Between January 2005 and February 2007, children with bacteriologically confirmed TB, children having contacts with a case of infectious pulmonary TB and children without any known risk for TB were tested with both the TST and T SPOT TB.</p> <p>Results</p> <p>The TST and T SPOT TB tests were positive for all patients in the „culture-confirmed TB“ group. Whereas, in the „high risk for TB“ group, the TST was positive for 60%, but the T SPOT TB test, only for 17.8%. Meanwhile the results for the „low risk for TB“ group were 65.4% and 9.6%, respectively. A correlation between the TST and T SPOT TB was obtained in the "culture-confirmed TB" group where the TST ≥15 mm (r = 0.35, p < 0.001).</p> <p>Conclusion</p> <p>The T-cell based method is more objective than the TST for identifying latent TB infection in children who had been previously BCG vaccinated. This method could be useful in countries like Lithuania where there is a high incidence of TB despite a high coverage with BCG vaccination. It may also help to avoid unnecessary chemoprophylaxis when TST reactions are false-positive.</p

Within-Subject Variability of Interferon-g Assay Results for Tuberculosis and Boosting Effect of Tuberculin Skin Testing: A Systematic Review

Background: Variability in interferon-gamma release assays (IGRAs) results for tuberculosis has implications for interpretation of results close to the cut-point, and for defining thresholds for test conversion and reversion. However, little is known about the within-subject variability (reproducibility) of IGRAs. Several national guidelines recommend a twostep testing procedure (tuberculin skin test [TST] followed by IGRA) for the diagnosis of LTBI. However, the effect of a preceding TST on subsequent IGRA results has been reported in studies with apparently conflicting results. Methodology/Findings: We conducted a systematic review to synthesize evidence on within-subject variability of IGRA results and the potential boosting effect of TST. We searched several databases and reviewed citations of previous reviews on IGRAs. We included studies using commercial IGRAs, in addition to non-commercial versions of the ELISPOT assay. Four studies, fulfilling our predefined criteria, examined within-subject variability and 13 studies evaluated TST effects on subsequent IGRA responses. Meta-analysis was not considered appropriate because of heterogeneity in study methods, assays, and populations. Although based on limited data, within-subject variability was present in all studies but the magnitude varied (16-80%) across studies. A TST induced ‘‘boosting’ ’ of IGRA responses was demonstrated in several studies and although more pronounced in IGRA-positive (i.e. sensitized) individuals, also occurred in a smaller but not insignificant proportion of IGRA-negative subjects. The TST appeared to affect IGRA responses only after 3 days and may apparentl

Comparison of two interferon gamma release assays in the diagnosis of Mycobacterium tuberculosis infection and disease in The Gambia

<p>Abstract</p> <p>Background</p> <p>IFN-γ Release Assays (IGRAs) have been licensed for the diagnosis of latent <it>Mycobacterium tuberculosis </it>infection (LTBI). Their performance may depend on assay format and may vary across populations and settings. We compared the diagnostic performance of an in-house T -cell and commercial whole blood-based IGRAs for the diagnosis of LTBI and TB disease in The Gambia.</p> <p>Methods</p> <p>Newly diagnosed sputum smear positive cases and their household contacts were recruited. Cases and contacts were bled for IGRA and contacts had a Mantoux skin test. We assessed agreement and discordance between the tests and categorized a contact's level of <it>M. tuberculosis </it>exposure according to where s/he slept relative to a case: the same room, same house or a different house. We assessed the relationship between exposure and test results by multiple logistic regression.</p> <p>Results</p> <p>In 80 newly diagnosed TB cases, the sensitivity of ELISPOT was 78.7% and for QFT-GIT was 64.0% (p = 0.047). Of 194 household contacts 57.1% and 58.8% were positive for ELISPOT and QFT-GIT respectively. The overall agreement between both IGRAs for LTBI in contacts was 71.4% and there was no significant discordance (p = 0.29). There was significant discordance between the IGRAs and TST. Neither IGRA nor TST had evidence of false positive results because of Bacille Calmette Guérin (BCG) vaccination. However, agreement between QFT-GIT and TST as well as discordance between both IGRAs and TST were associated with BCG vaccination. Both IGRAs responded to the <it>M. tuberculosis </it>exposure gradient and were positively associated with increasing TST induration (p = 0.003 for ELISPOT and p = 0.001 for QFT-GIT).</p> <p>Conclusion</p> <p>The ELISPOT test is more sensitive than the QFT-GIT for diagnosing TB disease. The two tests perform similarly in the diagnosis of LTBI in TB contacts. Significant discordance between the two IGRAs and between each and the TST remain largely unexplained.</p

A Three-Way Comparison of Tuberculin Skin Testing, QuantiFERON-TB Gold and T-SPOT.TB in Children

BACKGROUND: There are limited data comparing the performance of the two commercially available interferon gamma (IFN-gamma) release assays (IGRAs) for the diagnosis of tuberculosis (TB) in children. We compared QuantiFERON-TB gold In Tube (QFT-IT), T-SPOT.TB and the tuberculin skin test (TST) in children at risk for latent TB infection or TB disease. METHODS AND FINDINGS: The results of both IGRAs were compared with diagnosis assigned by TST-based criteria and assessed in relation to TB contact history. Results from the TST and at least one assay were available for 96 of 100 children. Agreement between QFT-IT and T-SPOT.TB was high (93% agreement, kappa = 0.83). QFT-IT and T-SPOT.TB tests were positive in 8 (89%) and 9 (100%) children with suspected active TB disease. There was moderate agreement between TST and either QFT-IT (75%, kappa = 0.50) or T-SPOT.TB (75%, kappa = 0.51). Among 38 children with TST-defined latent TB infection, QFT-IT gold and T-SPOT.TB assays were positive in 47% and 39% respectively. Three TST-negative children were positive by at least one IGRA. Children with a TB contact were more likely than children without a TB contact to have a positive IGRA (QFT-IT LR 3.9; T-SPOT.TB LR 3.9) and a positive TST (LR 1.4). Multivariate linear regression analysis showed that the magnitude of both TST induration and IGRA IFN-gamma responses was significantly influenced by TB contact history, but only the TST was influenced by age. CONCLUSIONS: Although a high level of agreement between the IGRAs was observed, they are commonly discordant with the TST. The correct interpretation of a negative assay in a child with a positive skin test in clinical practice remains challenging and highlights the need for longitudinal studies to determine the negative predictive value of IGRAs

Predictors of persistently positive Mycobacterium-tuberculosis-specific interferon-gamma responses in the serial testing of health care workers

<p>Abstract</p> <p>Background</p> <p>Data on the performance of Mycobacterium-tuberculosis-specific interferon-(IFN)-γ release assays (IGRAs) in the serial testing of health care workers (HCWs) is limited. The objective of the present study was to determine the frequency of IGRA conversions and reversions and to identify predictors of persistent IGRA positivity among serially tested German HCWs in the absence of recent extensive tuberculosis (TB) exposure.</p> <p>Methods</p> <p>In this observational cohort-study HCWs were prospectively recruited within occupational safety and health measures and underwent a tuberculin skin test (TST) and the IGRA QuantiFERON^®-TB Gold In-Tube (QFT-GIT) at baseline. The QFT-GIT was repeated 18 weeks later in the median. QFT-GIT conversions (and reversions) were defined as baseline IFN-γ < 0.35 IU/ml and follow-up IFN-γ ≥ 0.35 IU/ml (and vice versa). Predictors of persistently positive QFT-GIT results were calculated by logistic regression analysis.</p> <p>Results</p> <p>In total, 18 (9.9%) and 15 (8.2%) of 182 analyzed HCWs were QFT-GIT-positive at baseline and at follow-up, respectively. We observed a strong overall agreement between baseline and follow-up QFT-GIT results (κ = 0.70). Reversions (6/18, 33.3%) occurred more frequently than conversions (3/162, 1.9%). Age and positive prior and recent TST results independently predicted persistent QFT-GIT positivity. Furthermore, the chance of having persistently positive QFT-GIT results raised about 3% with each additional 0.1 IU/ml increase in the baseline IFN-γ response (adjusted odds ratio 1.03, 95% confidence interval 1.01-1.04). No active TB cases were detected within an observational period of more than two years.</p> <p>Conclusions</p> <p>The QFT-GIT's utility for the application in serial testing was limited by a substantial proportion of reversions. This shortcoming could be overcome by the implementation of a borderline zone for the interpretation of QFT-GIT results. However, further studies are needed to clearly define the within-subject variability of the QFT-GIT and to confirm that increasing age, concordantly positive TST results, and the extend of baseline IFN-γ responses may predict the persistence of QFT-GIT positivity over time in serially tested HCWs with only a low or medium TB screening risk in a TB low-incidence setting.</p

Hypoxia-Inducible Factor 1α Determines Gastric Cancer Chemosensitivity via Modulation of p53 and NF-κB

BACKGROUND: Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1alpha has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1alpha's role in determining chemosensitivity focussing on responsible molecular pathways. METHODOLOGY AND PRINCIPAL FINDINGS: RNA interference was applied to inactivate HIF-1alpha or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-kappaB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1alpha in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1alpha-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1alpha markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1alpha-competent cells was identified as the molecular mechanism of HIF-1alpha-mediated inhibition of p53. Furthermore, loss of HIF-1alpha abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-kappaB and expression of anti-apoptotic NF-kappaB target genes. Accordingly, reconstitution of the NF-kappaB subunit p65 reversed the increased chemosensitivity of HIF-1alpha-deficient cells. CONCLUSION AND SIGNIFICANCE: In summary, we identified HIF-1alpha as a potent regulator of p53 and NF-kappaB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy