Andrew R. DiNardo

Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children: a systematic review and meta-analysis.

BACKGROUND Microbiological confirmation of childhood tuberculosis is rare because of the difficulty of collection of specimens, low sensitivity of smear microscopy, and poor access to culture. We aimed to establish summary estimates for sensitivity and specificity of of the Xpert MTB/RIF assay compared with microscopy in the diagnosis of pulmonary tuberculosis in children. METHODS We searched for studies published up to Jan 6, 2015, that used Xpert in any setting in children with and without HIV infection. We systematically reviewed studies that compared the diagnostic accuracy of Xpert MTB/RIF (Xpert) with microscopy for detection of pulmonary tuberculosis and rifampicin resistance in children younger than 16 years against two reference standards-culture results and culture-negative children who were started on anti-tuberculosis therapy. We did meta-analyses using a bivariate random-effects model. FINDINGS We identified 15 studies including 4768 respiratory specimens in 3640 children investigated for pulmonary tuberculosis. Culture tests were positive for tuberculosis in 12% (420 of 3640) of all children assessed and Xpert was positive in 11% (406 of 3640). Compared with culture, the pooled sensitivities and specificities of Xpert for tuberculosis detection were 62% (95% credible interval 51-73) and 98% (97-99), respectively, with use of expectorated or induced sputum samples and 66% (51-81) and 98% (96-99), respectively, with use of samples from gastric lavage. Xpert sensitivity was 36-44% higher than was sensitivity for microscopy. Xpert sensitivity in culture-negative children started on antituberculosis therapy was 2% (1-3) for expectorated or induced sputum. Xperts pooled sensitivity and specificity to detect rifampicin resistance was 86% (95% credible interval 53-98) and 98% (94-100), respectively. INTERPRETATION Compared with microscopy, Xpert offers better sensitivity for the diagnosis of pulmonary tuberculosis in children and its scale-up will improve access to tuberculosis diagnostics for children. Although Xpert helps to provide rapid confirmation of disease, its sensitivity remains suboptimum compared with culture tests. A negative Xpert result does not rule out tuberculosis. Good clinical acumen is still needed to decide when to start antituberculosis therapy and continued research for better diagnostics is crucial. FUNDING WHO, Global TB Program of Texas Childrens Hospital.

Hookworm infection is associated with decreased CD4+T cell counts in HIV-infected adult Ugandans

Most studies evaluating epidemiologic relationships between helminths and HIV have been conducted in the pre-ART era, and evidence of the impact of helminth infections on HIV disease progression remains conflicting. Less is known about helminth infection and clinical outcomes in HIV-infected adults receiving antiretroviral therapy (ART). We sampled HIV-infected adults for eight gastrointestinal parasites and correlated parasitic infection with demographic predictors, and clinical and immunologic outcomes. Contrasting with previous studies, we measured parasitic infection with a quantitative, highly sensitive and specific polymerase chain reaction (PCR) method. This cohort study enrolled HIV-infected Ugandans from August-September 2013 in Mbale, Uganda and collected stool and blood samples at enrollment. Real-time PCR quantified stool: Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, Cryptosporidium spp., Entamoeba histolytica, and Giardia intestinalis infection. Generalized linear models assessed relationships between parasitic infection and clinical or demographic data. 35% of participants (71/202) tested positive for ≥1 helminth, mainly N. americanus (55/199, 28%), and 4.5% (9/202) were infected with ≥2 stool parasites. Participants with hookworm infection had lower average CD4+ cell counts (-94 cells/mcL, 95%CI: -141, -48 cells/mcL; p<0.001) after adjustment for sex, CD4+ nadir at clinic entry, and time on ART. The high prevalence of parasitic infection and correlation with decreased CD4+ concentrations highlight the need to re-examine the effects of invasive helminth co-infection in rural, HIV-infected populations in the era of widely available ART. Elucidating the relationship between hookworm infection and immune recovery could provide opportunities for health optimization, e.g. integrated deworming, in these vulnerable populations.

Schistosome Soluble Egg Antigen Decreases Mycobacterium tuberculosis–Specific CD4+ T-Cell Effector Function With Concomitant Arrest of Macrophage Phago-Lysosome Maturation

Helminth-infected individuals possess a higher risk of developing tuberculosis, but the precise immunologic mechanism of Mycobacterium tuberculosis control remains unclear. We hypothesized that a perturbation of the M. tuberculosis-specific CD4(+) T-cell response weakens the ability of macrophages to contain M. tuberculosis We exposed peripheral blood mononuclear cells from M. tuberculosis-infected humans to schistosome soluble egg antigen (SEA) and then profiled M. tuberculosis-specific CD4(+) T cells via multiparametric flow cytometry. SEA decreased the frequency of cells producing interferon γ (6.79% vs 3.20%; P = .017) and tumor necrosis factor α (6.98% vs 2.96%; P = .012), with a concomitant increase in the median fluorescence intensity of interleukin 4 (IL-4; P < .05) and interleukin 10 (IL-10; 1440 vs 1273; P < .05). Macrophages polarized with SEA-exposed, autologous CD4(+) T-cell supernatant had a 2.19-fold decreased colocalization of lysosomes and M. tuberculosis (P < .05). When polarized with IL-4 or IL-10, macrophages had increased expression of CD206 (P < .0001), 1.5-fold and 1.9 fold increased intracellular numbers of M. tuberculosis per macrophage (P < .0005), and 1.4-fold and 1.7-fold decreased colocalization between M. tuberculosis and lysosomes (P < .001). This clarifies a relationship in which helminth-induced CD4(+) T cells disrupt M. tuberculosis control by macrophages, thereby providing a mechanism for the observation that helminth infection advances the progression of tuberculosis among patients with M. tuberculosis infection.

Why being an expert – despite xpert –remains crucial for children in high TB burden settings

BackgroundAs access to Xpert expands in high TB-burden settings, its performance against clinically diagnosed TB as a reference standard provides important insight as the majority of childhood TB is bacteriologically unconfirmed. We aim to describe the characteristics and outcomes of children with presumptive TB and TB disease, and assess performance of Xpert under programmatic conditions against a clinical diagnosis of TB as a reference standard.MethodsRetrospective review of children evaluated for presumptive TB in Mbeya, Tanzania. Baseline characteristics were compared by TB disease status and, for patients diagnosed with TB, by TB confirmation status using Wilcoxon rank sum test for continuous variables and the Chi-square test for categorical variables. Sensitivity and specificity were calculated to assess the performance of Xpert, smear, and culture against clinical TB. Kappa statistics were calculated to assess agreement between Xpert and smear to culture.ResultsAmong children (N = 455) evaluated for presumptive TB, 70.3% (320/455) had Xpert and 62.8% (286/455) had culture performed on sputa. 34.5% (157/455) were diagnosed with TB: 80.3% (126/157) pulmonary TB, 13.4% (21/157) bacteriologically confirmed, 53.5% (84/157) HIV positive, and 48.4% (76/157) inpatients. Compared to the reference standard of clinical diagnosis, sensitivity of Xpert was 8% (95% CI 4–15), smear 6% (95% CI 3–12) and culture 16% (95% CI 9–24), and did not differ based on patient disposition, nutrition or HIV status.ConclusionDespite access to Xpert, the majority of children with presumptive TB were treated based on clinical diagnosis. Reflecting the reality of clinical practice in resource limited settings, new diagnostics such as Xpert serve as important adjunctive tests but will not obviate the need for astute clinicians and comprehensive diagnostic algorithms.

Use of string test and stool specimens to diagnose pulmonary tuberculosis

SUMMARY Background The Xpert MTB/RIF (MTB/RIF) test has advanced the field of tuberculosis (TB) diagnostics; however, depending on age and HIV status, 10–85% of individuals with presumed pulmonary TB (PTB) are unable to produce sputum. Methods The feasibility of using MTB/RIF and culture on stool and string test specimens from 13 adult patients with presumed PTB was studied. Results The string test was well tolerated with a median Wong Baker Faces score of 2. The string test had 100% sensitivity and specificity by MTB/RIF and 87.5% sensitivity and 100% specificity by culture. In stool, Mycobacterium tuberculosis DNA was detected in all cases of culture-confirmed PTB. Conclusion The string test and stool provide diagnostic specimens that warrant further investigation.

HIV Progression Perturbs the Balance of the Cell-Mediated and Anti-Inflammatory Adaptive and Innate Mycobacterial Immune Response

Introduction. Our objective is to understand how HIV infection increases the risk of progression from latent tuberculosis (TB) to active disease. We understand now that immunity is a balance of competing immune responses by multiple cell types. Since T-lymphocyte production of interferon-gamma (IFN-γ) in response to Mycobacterium tuberculosis (Mtb) antigens fails to differentiate disease from latent infection, we applied a comprehensive profiling methodology to define immune biomarkers that reliably predict a patients TB risk. Methods. We established a cohort of HIV-infected adults with TB disease from Swaziland. Multiparametric flow cytometry was used to quantify the mycobacterial-specific anti-inflammatory (IL-4 and IL-10) and proinflammatory (IFN-γ) immune response. Results. From 12 HIV-infected Swaziland patients with TB disease, the CD4+, CD8+, Double Negative, and CD56+CD3− lymphocytes increase their IL-4 : IFN-γ ratio as HIV disease worsens (Spearman r of −0.59; −0.59; −0.60; and −0.59, resp.; p < 0.05). Similarly, HIV severity is associated with an increased IL-10 : IFN-γ ratio (Spearman r of −0.76; p = 0.01). Conclusion. As HIV disease progresses, both the adaptive and innate branches skew away from an inflammatory and towards anti-inflammatory phenotype.

Paradoxical immune reconstitution inflammatory syndrome due to toxoplasmic encephalitis: two cases and review of initiation of antiretroviral timing in toxoplasmic encephalitis IRIS

Toxoplasma encephalitis immune reconstitution inflammatory syndrome (TE-IRIS) is rare and usually occurs in an unmasking, rather than paradoxical form. To the best of our knowledge, only two cases of paradoxical TE-IRIS and nine cases of unmasking TE-IRIS have been previously described. We present two additional cases of histopathology-consistent paradoxical TE-IRIS, after early initiation of antiretroviral therapy (ART), and review the literature on TE-IRIS. Three of the four reported cases of paradoxical TE-IRIS were associated with early (within one week) initiation of ART, an issue that was not addressed in the 2009 US Department of Health and Human Services guidelines for the treatment of opportunistic infections.

Schistosomiasis Induces Persistent DNA Methylation and Tuberculosis-Specific Immune Changes

Epigenetic mechanisms, such as DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth coinfections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)–specific and bacillus Calmette–Guérin–induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared with age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA-methylated genes, with 72% hypermethylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted real-time quantitative PCR after methyl-specific endonuclease digestion confirmed DNA hypermethylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4, and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2, and IL12B (p < 0.001; Sidak–Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed, with helminth-infected individuals having decreased upregulation of IFN-γ–inducible genes (Mann–Whitney p < 0.05). Hypomethylation of the IL-4 pathway and DNA hypermethylation of the Th1 pathway was confirmed by Ag-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed-rank p < 0.05). In S. haematobium–infected individuals, these DNA methylation and immune phenotypic changes persisted at least 6 mo after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth specific.

Diagnostic and Treatment Monitoring Potential of A Stool-Based Quantitative Polymerase Chain Reaction Assay for Pulmonary Tuberculosis

A quantifiable, stool-based, Mycobacterium tuberculosis (Mtb) test has potential complementary value to respiratory specimens. Limit of detection (LOD) was determined by spiking control stool. Clinical test performance was evaluated in a cohort with pulmonary tuberculosis (TB) (N = 166) and asymptomatic household TB child contacts (N = 105). Stool-quantitative polymerase chain reaction (qPCR) results were compared with sputum acid-fast bacilli (AFB) microscopy, GeneXpert MTB/RIF (Xpert MTB/RIF), and cultures. In Mtb stool-spiking studies, the LOD was 96 colony-forming units/50 mg of stool (95% confidence interval [CI]: 84.8-105.6). Among specimens collected within 72 hours of antituberculosis treatment (ATT) initiation, stool qPCR detected 22 of 23 (95%) of culture-positive cases. Among clinically diagnosed cases that were Xpert MTB/RIF and culture negative, stool qPCR detected an additional 8% (3/37). Among asymptomatic, recently TB-exposed participants, stool PCR detected Mtb in two of 105 (1.9%) patients. Two months after ATT, the Mtb quantitative burden in femtogram per microliters decreased (Wilcoxon signed-rank P < 0.001) and persistent positive stool PCR was associated with treatment failure or drug resistance (relative risk 2.8, CI: 1.2-6.5; P = 0.012). Stool-based qPCR is a promising complementary technique to sputum-based diagnosis. It detects and quantifies low levels of stool Mtb DNA, thereby supporting adjunct diagnosis and treatment monitoring in pulmonary TB.

Reactivation tuberculosis: role of surveillance

ABSTRACT The incidence and death rates from tuberculosis (TB) have declined through concerted efforts in the diagnosis and treatment of active disease. Despite this, 9.6 million new cases and 1.1 million deaths in 2014 are unacceptably high. To decrease the rates of TB further, the huge number of persons with latent TB infection (LTBI) from whom new cases will arise has to be addressed with a sense of priority. Identifying the highest risk groups and providing effective treatment has been shown to decrease active TB. Further research to refine the predictors of reactivation and shorter effective treatments are urgently needed. Implementing intensified case finding, testing and treatment for LTBI will require continued investment in health care capacity at multiple levels.