The successful treatment of tuberculosis (TB) requires long-term multidrug chemotherapy. Clinical trials to evaluate new drugs and regimens for TB treatment are protracted due to the slow clearance of Mycobacterium tuberculosis (Mtb) infection and the lack of early biomarkers to predict treatment outcome. Advancements in the field of metabolomics make it possible to identify metabolic profiles that correlate with disease states or successful chemotherapy. However, proof-of-concept of this approach has not been provided for a TB-early treatment response biosignature (TB-ETRB).
Urine samples collected at baseline and during treatment from 48 Ugandan and 39 South African HIV-seronegative adults with pulmonary TB were divided into discovery and qualification sets, normalized to creatinine concentration, and analyzed by liquid chromatography-mass spectrometry to identify small molecule molecular features (MFs) in individual patient samples. A biosignature that distinguished baseline and 1 month treatment samples was selected by pairwise t-test using data from two discovery sample sets. Hierarchical clustering and repeated measures analysis were applied to additional sample data to down select molecular features that behaved consistently between the two clinical sites and these were evaluated by logistic regression analysis.
Analysis of discovery samples identified 45 MFs that significantly changed in abundance at one month of treatment. Down selection using an extended set of discovery samples and qualification samples confirmed 23 MFs that consistently changed in abundance between baseline and 1, 2 and 6 months of therapy, with 12 MFs achieving statistical significance (p < 0.05). Six MFs classified the baseline and 1 month samples with an error rate of 11.8%.
These results define a urine based TB-early treatment response biosignature (TB-ETRB) applicable to different parts of Africa, and provide proof-of-concept for further evaluation of this technology in monitoring clinical responses to TB therapy.
Tuberculosis; Metabolomics; Biomarker; Mass spectrometry; Small molecule biosignature; Anti-tuberculosis therapy; Mycobacterium tuberculosis, Urine
The global epidemiology of parasitic helminths and mycobacterial infections display extensive geographical overlap, especially in the rural and urban communities of developing countries. We investigated whether co-infection with the gastrointestinal tract-restricted helminth, Trichuris muris, and the intracellular bacterium, Mycobacterium bovis (M. bovis) BCG, would alter host immune responses to, or the pathological effect of, either infection.
We demonstrate that both pathogens are capable of negatively affecting local and systemic immune responses towards each other by modifying cytokine phenotypes and by inducing general immune suppression. T. muris infection influenced non-specific and pathogen-specific immunity to M. bovis BCG by down-regulating pulmonary TH1 and Treg responses and inducing systemic TH2 responses. However, co-infection did not alter mycobacterial multiplication or dissemination and host pulmonary histopathology remained unaffected compared to BCG-only infected mice. Interestingly, prior M. bovis BCG infection significantly delayed helminth clearance and increased intestinal crypt cell proliferation in BALB/c mice. This was accompanied by a significant reduction in systemic helminth-specific TH1 and TH2 cytokine responses and significantly reduced local TH1 and TH2 responses in comparison to T. muris-only infected mice.
Our data demonstrate that co-infection with pathogens inducing opposing immune phenotypes, can have differential effects on compartmentalized host immune protection to either pathogen. In spite of local and systemic decreases in TH1 and increases in TH2 responses co-infected mice clear M. bovis BCG at the same rate as BCG only infected animals, whereas prior mycobacterial infection initiates prolonged worm infestation in parallel to decreased pathogen-specific TH2 cytokine production.
Helminth; Co-infection; Mycobacteria; Tuberculosis
The diagnosis of tuberculosis remains challenging in individuals with difficulty in providing good quality sputum samples such as children. Host biosignatures of inflammatory markers may be valuable in such cases, especially if they are based on more easily obtainable samples such as saliva. To explore the potential of saliva as an alternative sample in tuberculosis diagnostic/biomarker investigations, we evaluated the levels of 33 host markers in saliva samples from individuals presenting with pulmonary tuberculosis symptoms and compared them to those obtained in serum. Of the 38 individuals included in the study, tuberculosis disease was confirmed in 11 (28.9%) by sputum culture. In both the tuberculosis cases and noncases, the levels of most markers were above the minimum detectable limit in both sample types, but there was no consistent pattern regarding the ratio of markers in serum/saliva. Fractalkine, IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF, and IL-5 levels in saliva and IL-6, IL-2, SAP, and SAA levels in serum were significantly higher in tuberculosis patients (P < 0.05). These preliminary data indicate that there are significant differences in the levels of host markers expressed in saliva in comparison to those expressed in serum and that inflammatory markers in both sample types are potential diagnostic candidates for tuberculosis disease.
The contraceptive depot medroxyprogesterone acetate (DMPA), with progestin as the single active compound, possesses selective glucocorticoid activity and can alter the expression of glucocorticoid receptor-regulated genes. We therefore propose that pharmacological doses of DMPA used for endocrine therapy could have significant immune modulatory effects and impact on susceptibility to, as well as clinical manifestation and outcome of, infectious diseases. We investigated the effect of contraceptive doses of DMPA in two different murine Mycobacterium tuberculosis models. Multiplex bead array analysis revealed that DMPA altered serum cytokine levels of tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), and interleukin 10 (IL-10) in C57BL/6 mice and gamma interferon (IFN-γ) in BALB/c mice. DMPA also suppressed antigen-specific production of TNF-α, G-CSF, IL-10, and IL-6 and induced the production of IP-10 in C57BL/6 mice. In BALB/c mice, DMPA altered the antigen-specific secretion of IFN-γ, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and monocyte chemotactic protein 1 (MCP-1). Furthermore, we show that C57BL/6 mice treated with doses of DMPA, which result in serum concentrations similar to those observed in contraceptive users, have a significantly higher bacterial load in their lungs. Our data show for the first time that DMPA impacts tuberculosis (TB) disease severity in a mouse model and that the effects of this contraceptive are not confined to infections of the genital tract. This could have major implications for the contraceptive policies not only in developing countries like South Africa but also worldwide.
Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas.
We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens.
There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST- and TST+ contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST+ contacts (LTBI) compared to TB and TST- contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen.
Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.
Background. Diagnosis and treatment of sexually transmitted infections (STIs) is a public health priority, particularly in regions where the incidence of human immunodeficiency virus (HIV) infection is high. In most developing countries, STIs are managed syndromically. We assessed the adequacy of syndromic diagnosis of STIs, compared with laboratory diagnosis of STIs, and evaluated the association between STI diagnosis and the risk of HIV acquisition in a cohort of high-risk women.
Methods. HIV-uninfected high-risk women (n = 242) were followed for 24 months. Symptoms of STIs were recorded, and laboratory diagnosis of common STI pathogens was conducted every 6 months. Forty-two cytokines were measured by Luminex in cervicovaginal lavage specimens at enrollment. Human immunodeficiency virus type 1 (HIV-1) infection was evaluated monthly.
Results. Only 12.3% of women (25 of 204) who had a laboratory-diagnosed, discharge-causing STI had clinically evident discharge. Vaginal discharge was thus a poor predictor of laboratory-diagnosed STIs (sensitivity, 12.3%; specificity, 93.8%). Cervicovaginal cytokine concentrations did not differ between women with asymptomatic STIs and those with symptomatic STIs and were elevated in women with asymptomatic STIs, compared with women with no STIs or bacterial vaginosis. Although laboratory-diagnosed STIs were associated with increased risk of HIV infection (hazard ratio, 3.3 [95% confidence interval, 1.5–7.2)], clinical symptoms were not.
Conclusions. Syndromic STI diagnosis dependent on vaginal discharge was poorly predictive of laboratory-diagnosed STI. Laboratory-diagnosed STIs were associated with increased susceptibility to HIV acquisition, while vaginal discharge was not.
The diagnosis of childhood tuberculosis (TB) disease remains a challenge especially in young and HIV-infected children. Recent studies have identified potential host markers which, when measured in Quantiferon (QFT-IT) supernatants, show promise in discriminating between Mycobacterium tuberculosis (M.tb) infection states. In this study, the utility of such markers was investigated in children screened for TB in a setting with high TB incidence.
Methodology and Principal Findings
76 children (29% HIV-infected) with or without active TB provided blood specimens collected directly into QFT-IT tubes. After overnight incubation, culture supernatants were harvested, aliquoted and frozen for future immunological research purposes. Subsequently, the levels of 12 host markers previously identified as potential TB diagnostic markers were evaluated in these supernatants for their ability to discriminate between M.tb infection and disease states using the Luminex platform. Of the 76 children included, 19 (25%) had culture confirmed TB disease; 26 (46%) of the 57 without TB had positive markers of M.tb infection defined by a positive QFT-IT test. The potentially most useful analytes for diagnosing TB disease included IFN-α2, IL-1Ra, sCD40L and VEGF and the most useful markers for discriminating between QFT-IT positive children as TB or latent infection included IL-1Ra, IP-10 and VEGF. When markers were used in combinations of four, 84% of all children were accurately classified into their respective groups (TB disease or no TB), after leave-one-out cross validation.
Measurement of the levels of IFN-α2, IL-1Ra, sCD40L, IP-10 and VEGF in QFT-IT supernatants may be a useful method for diagnosing TB disease and differentiating between active TB disease and M.tb infection in children. Our observations warrant further investigation in larger well-characterized clinical cohorts.
The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4+ T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4+ T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease.
We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.
Background. The biggest challenge in human immunodeficiency virus type 1 (HIV-1) prevention in Africa is the high HIV-1 burden in young women. In macaques, proinflammatory cytokine production in the genital tract is necessary for target cell recruitment and establishment of simian immunodeficiency virus (SIV) infection following vaginal inoculation. The purpose of this study was to assess if genital inflammation during early HIV-1 infection predisposes women to rapid disease progression.
Methods. Inflammatory cytokine concentrations were measured in cervicovaginal lavage (CVL) from 49 women 6, 17, 30, and 55 weeks after HIV-1 infection and from 22 of these women before infection. Associations between genital inflammation and viral load set point and blood CD4 cell counts 12 months after infection were investigated.
Results. Elevated genital cytokine concentrations 6 and 17 weeks after HIV-1 infection were associated with higher viral load set points and, to a lesser extent, with CD4 depletion. CVL cytokine concentrations during early infection did not differ relative to preinfection but were elevated in women who had vaginal discharge, detectable HIV-1 RNA in their genital tracts, and lower blood CD4 counts.
Conclusion. Genital inflammation during early HIV-1 infection was associated with higher viral load set point and CD4 depletion, which are markers of rapid disease progression. Strategies aimed at reducing genital inflammation during early HIV-1 infection may slow disease progression.
Understanding the genetic diversity of Mycobacterium tuberculosis is needed for a better understanding of the epidemiology of TB and could have implications for the development of new diagnostics, drugs, and vaccines. M. tuberculosis isolates were characterized using spoligotyping and were compared with the SpoIDB4 database of the Pasteur Institute of Guadeloupe. A total of 53 different patterns were identified among 192 isolates examined. 169 of the isolates were classified into one of the 33 shared SITs, whereas the remaining 23 corresponded to 20 orphan patterns. 54% of the isolates were ascribed to the T family, a family which has not been well defined to date. Other prominent families were CAS, Haarlem, LAM, Beijing, and Unknown comprising 26%, 13%, 2.6%, 0.5%, and 2.1%, respectively. Among HIV-positive patients, 10 patterns were observed among 25 isolates. The T (38.5%), H (26.9%), and CAS (23.1%) families were the most common among HIV-positive individuals. The diversity of the M. tuberculosis strains found in this study is very high, and there was no difference in the distribution of families in HIV-positive and HIV-negative TB patients except the H family. Tuberculosis transmission in Addis Ababa is due to only the modern M. tuberculosis families (CAS, LAM, T, Beijing, Haarlem, and U).
The mycobacterial heparin-binding hemagglutinin (HBHA) protein induces a potent gamma interferon (IFN-γ) response in latent tuberculosis (TB) infection and is a candidate vaccine and diagnostic antigen. We have assessed HBHA-specific intracellular IFN-γ, interleukin-2 (IL-2), and IL-17 production by CD4+ T cells in TB cases and household contacts (HHCs) as well as the level of secreted IFN-γ in whole-blood culture supernatant. HHCs were further classified as tuberculin skin test (TST) positive or negative, and the group was also divided as HIV positive or negative. Our study revealed that HBHA induces multifunctional IFN-γ-, IL-2-, and IL-17-coexpressing CD4+ T cells in HHCs but not in active TB cases; however, IFN-γ levels in culture supernatant did not differ between participant groups. Further studies are needed to completely understand how HBHA induces immune responses in different disease groups.
Epidemiological evidence from tuberculosis outbreaks revealed that some genotypes of M. tuberculosis are more transmissible and capable of causing disease than others. We analysed the plasma cytokine levels of pulmonary tuberculosis patients infected with different strains of M. tuberculosis to test the hypothesis that immune responses would be linked to the bacterial genotype. Spoligotyping was carried out for genotyping, and we used Luminex technology to measure 17 cytokines (EGF, fractalkine, GM-CSF, IFN-γ, IL-1, IL-10, IL-12, IL-17, IL-4, IL-7, IL-9, IP-10, MCP-1, MCP-3, MIP-1β, TNF, and VEGF) from plasma samples of tuberculosis patients. The levels of IL-12 (p40), IL-4, IL-7, and MIP-1beta were higher in patients infected with lineage 3, however, it was only IL-4 which showed statistically significant difference (P < 0.05) between lineage 3 and lineage 4. We further grouped the lineages into families (CAS, H and T families), and we found that the plasma level of IL-4 was significantly higher in patients infected with the CAS family (P < 0.05) in comparison with T and H families. However, there was no difference between T and H families. Therefore, the higher level of IL-4 in lineage 3 families might indicate that possible differences in the response elicited from host depend on strain lineages in the studied population.
M.tuberculosis (M.tb) is associated with enhanced T helper cell type 1 (Th1) immune responses while helminth infection is associated with T helper cell type 2 (Th2) immune responses. Our aim was to investigate whether helminth infection could influence the ability to generate an appropriate Th1 immune response that is characterized by a positive tuberculin skin test (TST), in M.tb exposed children.
We completed a community-based, cross sectional household contact tracing study, using matched enrolment of HIV negative children with and without documented household M.tb exposure. We documented demographics, clinical characteristics, HIV status, M.tb exposure (using a standard contact score) and M.tb infection status (TST > = 10 mm). Ascaris lumbricoides-specific IgE was used as proxy for Ascaris infection/exposure.
Of 271 children (median age 4 years (range: 4 months to 15 years)) enrolled, 65 participants (24%) were serum positive for Ascaris IgE. There were 168 (62%) children with a documented household tuberculosis contact and 107 (40%) were (TST) positive overall.
A positive TST was associated with increasing age (Odds Ratio (OR) =1.17, p < 0.001), increasing M.tb contact score (OR = 1.17, p < 0.001), previous tuberculosis treatment (OR = 4.8, p = 0.06) and previous isoniazid preventive treatment (OR = 3.16, p = 0.01). A visible bacillus Calmette-Guérin (BCG) scar was associated with reduced odds of being TST positive (OR = 0.42, p = 0.01).
Ascaris IgE was not associated with TST status in univariate analysis (OR = 0.9, p = 0.6), but multivariable logistic regression analysis suggested an inverse association between Ascaris IgE status and a positive TST (OR = 0.6, p = 0.08), when adjusted for age, and M.tb contact score. The addition of an age interaction term to the model suggested that the age effect was stronger among Ascaris IgE positive children; the effect of being Ascaris IgE positive significantly reduced the odds of being TST positive amongst younger children while this effect weakened with increasing age.
Our preliminary findings highlight a high prevalence of both Ascaris exposure/infection and M.tb infection in children in an urban setting. Helminth exposure/infection may reduce the immune response following M.tb exposure when controlling for epidemiological and clinical covariates. These findings might be relevant to the interpretation of immunological tests of M.tb infection in children.
Tuberculosis; Helminth infection; Ascaris, M.tb infection; Immune polarization; Paediatric tuberculosis
One third of the world’s population is infected with Mycobacterium tuberculosis (M.tb). A vaccine that would prevent progression to TB disease will have a dramatic impact on the global TB burden. We propose that antigens of M.tb that are preferentially expressed during latent infection will be excellent candidates for post-exposure vaccination. We therefore assessed human T cell recognition of two such antigens, Rv2660 and Rv2659. Expression of these was shown to be associated with non-replicating persistence in vitro. After six days incubation of PBMC from persons with latent tuberculosis infection (LTBI) and tuberculosis (TB) disease, Rv2660 and Rv2659 induced IFN-γ production in a greater proportion of persons with LTBI, compared with TB diseased patients. Persons with LTBI also had increased numbers of viable T cells, and greater specific CD4+ T cell proliferation and cytokine expression capacity. Persons with LTBI preferentially recognize Rv2659 and Rv2660, compared with patients with TB disease. These results suggest promise of these antigens for incorporation into post-exposure TB vaccines.
Mycobacterium tuberculosis; LTBI; TB disease; latency antigens; post-infection vaccine
Recent interferon gamma (IFN-γ)-based studies have identified novel Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens as diagnostic candidates. In this study, the levels of 11 host markers other than IFN-γ, were evaluated in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens, for the diagnosis of TB disease.
Methodology and Principal Findings
Five M.tb infection phase-dependent antigens, comprising of three DosR-regulon-encoded proteins (Rv2032, Rv0081, Rv1737c), and two resucitation promoting factors (Rv0867c and Rv2389c), were evaluated in a case-control study with 15 pulmonary TB patients and 15 household contacts that were recruited from a high TB incidence setting in Cape Town, South Africa. After a 7-day whole blood culture, supernatants were harvested and the levels of the host markers evaluated using the Luminex platform. Multiple antigen-specific host markers were identified with promising diagnostic potential. Rv0081-specific levels of IL-12(p40), IP-10, IL-10 and TNF-α were the most promising diagnostic candidates, each ascertaining TB disease with an accuracy of 100%, 95% confidence interval for the area under the receiver operating characteristics plots, (1.0 to 1.0).
Multiple cytokines other than IFN-γ in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens show promise as diagnostic markers for active TB. These preliminary findings should be verified in well-designed diagnostic studies employing short-term culture assays.
Sputum Mycobacterium tuberculosis (Mtb) culture is commonly used to assess response to antibiotic treatment in individuals with pulmonary tuberculosis (TB). Such techniques are constrained by the slow growth rate of Mtb, and more sensitive methods to monitor Mtb clearance are needed. The goal of this study was to evaluate changes in plasma cytokines in patients undergoing treatment for TB as a means of identifying candidate host markers associated with microbiologic response to therapy.
Twenty-four plasma cytokines/chemokines were measured in 42 individuals diagnosed with active pulmonary TB, 52% were HIV co-infected. Individuals, undergoing a 26-week standard TB treatment, were followed longitudinally over 18 months and measurements were associated with HIV status and rates of sputum culture conversion.
Plasma concentrations of interferon-inducible protein-10 (IP-10) and vascular endothelial growth factor (VEGF) were significantly reduced upon TB treatment, regardless of HIV status. By the end of treatment, IP-10 concentrations were significantly lower in HIV negative individuals when compared to HIV-positive individuals (p = 0.02). Moreover, in HIV negative patients, plasma VEGF concentrations, measured as early as 2-weeks post TB treatment initiation, positively correlated with the time of sputum conversion (p = 0.0017). No significant changes were observed in other studied immune mediators.
These data suggest that VEGF plasma concentration, measured during early TB treatment, could represent a surrogate marker to monitor sputum culture conversion in HIV uninfected individuals.
A molecular assay to quantify Mycobacterium tuberculosis is described. In vitro, 98% (n = 96) of sputum samples with a known number of bacilli (107 to 102 bacilli) could be enumerated within 0.5 log10. In comparison to culture, the molecular bacterial load (MBL) assay is unaffected by other microorganisms present in the sample, results are obtained more quickly (within 24 h) and are seldom inhibited (0.7% samples), and the MBL assay critically shows the same biphasic decline as observed longitudinally during treatment. As a biomarker of treatment response, the MBL assay responds rapidly, with a mean decline in bacterial load for 111 subjects of 0.99 log10 (95% confidence interval [95% CI], 0.81 to 1.17) after 3 days of chemotherapy. There was a significant association between the rate of bacterial decline during the same 3 days and bacilli ml−1 sputum at day 0 (linear regression, P = 0.0003) and a 3.62 increased odds ratio of relapse for every 1 log10 increase in pretreatment bacterial load (95% CI, 1.53 to 8.59).
Confirming tuberculosis (TB) disease in suspects in resource limited settings is challenging and calls for the development of more suitable diagnostic tools. Different Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens may be differentially recognized in infected and diseased individuals and therefore useful as diagnostic tools for differentiating between M.tb infection states. In this study, we assessed the diagnostic potential of 118 different M.tb infection phase-dependent antigens in TB patients and household contacts (HHCs) in a high-burden setting.
Antigens were evaluated using the 7-day whole blood culture technique in 23 pulmonary TB patients and in 19 to 21 HHCs (total n = 101), who were recruited from a high-TB incidence community in Cape Town, South Africa. Interferon-gamma (IFN-γ) levels in culture supernatants were determined by ELISA.
Eight classical TB vaccine candidate antigens, 51 DosR regulon encoded antigens, 23 TB reactivation antigens, 5 TB resuscitation promoting factors (rpfs), 6 starvation and 24 other stress response-associated TB antigens were evaluated in the study. The most promising antigens for ascertaining active TB were the rpfs (Rv0867c, Rv2389c, Rv2450c, Rv1009 and Rv1884c), with Areas under the receiver operating characteristics curves (AUCs) between 0.72 and 0.80. A combination of M.tb specific ESAT-6/CFP-10 fusion protein, Rv2624c and Rv0867c accurately predicted 73% of the TB patients and 80% of the non-TB cases after cross validation.
IFN-γ responses to TB rpfs show promise as TB diagnostic candidates and should be evaluated further for discrimination between M.tb infection states.
Time to detection (TTD) on automated liquid mycobacterial cultures is an emerging biomarker of tuberculosis outcomes. The M. tuberculosis W-Beijing genotype is spreading globally, indicating a selective advantage. There is a paucity of data on the association between baseline TTD and W-Beijing genotype and tuberculosis outcomes.
To assess baseline predictors of failure of sputum culture conversion, within the first 2 months of antitubercular therapy, in participants with pulmonary tuberculosis.
Between May 2005 and August 2008 we conducted a prospective cohort study of time to sputum culture conversion in ambulatory participants with first episodes of smear and culture positive pulmonary tuberculosis attending two primary care clinics in Cape Town, South Africa. Rifampicin resistance (diagnosed on phenotypic susceptibility testing) was an exclusion criterion. Sputum was collected weekly for 8 weeks for mycobacterial culture on liquid media (BACTEC MGIT 960). Due to missing data, multiple imputation was performed. Time to sputum culture conversion was analysed using a Cox-proportional hazards model. Bayesian model averaging determined the posterior effect probability for each variable.
113 participants were enrolled (30.1% female, 10.5% HIV-infected, 44.2% W-Beijing genotype, and 89% cavities). On Kaplan Meier analysis 50.4% of participants underwent sputum culture conversion by 8 weeks. The following baseline factors were associated with slower sputum culture conversion: TTD (adjusted hazard ratio (aHR) = 1.11, 95% CI 1.02; 1.2), lung cavities (aHR = 0.13, 95% CI 0.02; 0.95), ever smoking (aHR = 0.32, 95% CI 0.1; 1.02) and the W-Beijing genotype (aHR = 0.51, 95% CI 0.25; 1.07). On Bayesian model averaging, posterior probability effects were strong for TTD, lung cavitation and smoking and moderate for W-Beijing genotype.
We found that baseline TTD, smoking, cavities and W-Beijing genotype were associated with delayed 2 month sputum culture. Larger studies are needed to confirm the relationship between the W-Beijing genotype and sputum culture conversion.
Increased access to combination antiretroviral therapy in areas co-endemic for tuberculosis (TB) and HIV-1 infection is associated with an increased incidence of immune reconstitution inflammatory syndrome (TB-IRIS) whose cause is poorly understood.
A case-control analysis of pro- and anti-inflammatory cytokines in TB-IRIS patients sampled at clinical presentation, and similar control patients with HIV-TB prescribed cART who did not develop TB-IRIS. Peripheral blood mononuclear cells were cultured in the presence or absence of heat killed M. tuberculosis (MTB) for 6 and 24 hours.
Stimulation with MTB increased the abundance of many cytokine transcripts with IL-1 Beta, IL-5, IL-6, IL-10, IL-13, IL-17A, IFN-gamma, GM-CSF and TNF being greater in stimulated TB-IRIS cultures. Analysis of the corresponding proteins in culture supernatants, revealed increased IL-1Beta, IL-2, IL-6, IL-8, IL-10, IL-12p40, IFN gamma, GM-CSF and TNF in TB-IRIS cultures. In serum, higher concentrations of TNF, IL-6, and IFN-gamma were observed in TB-IRIS patients̃ Serum IL-6 and TNF decreased during prednisone therapy in TB-IRIS patients.
These data suggest that cytokine release contributes to pathology in TB-IRIS. IL-6 and TNF were consistently elevated and decreased in serum during corticosteroid therapy. Specific blockade of these cytokines may be rational approach to immunomodulation in TB-IRIS.
HIV-1; Immune reconstitution inflammatory syndrome; Immunology; Tuberculosis
The inflammatory response to Mycobacterium tuberculosis (M.tb) at the site of disease is Th1 driven. Whether the Th17 cytokines, IL-17 and IL-22, contribute to this response in humans is unknown. We hypothesized that IL-17 and IL-22 contribute to the inflammatory response in pleural and pericardial disease sites of human tuberculosis (TB).
We studied pleural and pericardial effusions, established TB disease sites, from HIV-uninfected TB patients. Levels of soluble cytokines were measured by ELISA and MMP-9 by luminex. Bronchoalveolar lavage or pericardial mycobacteria-specific T cell cytokine expression was analyzed by intracellular cytokine staining.
IL-17 was not abundant in pleural or pericardial fluid. IL-17 expression by mycobacteria-specific disease site T cells was not detected in healthy, M.tb-infected persons, or patients with TB pericarditis. These data do not support a major role for IL-17 at established TB disease sites in humans.
IL-22 was readily detected in fluid from both disease sites. These IL-22 levels exceeded matching peripheral blood levels. Further, IL-22 levels in pericardial fluid correlated positively with MMP-9, an enzyme known to degrade the pulmonary extracellular matrix. We propose that our findings support a role for IL-22 in TB-induced pathology or the resulting repair process.
Pleural tuberculosis; Pericardial tuberculosis; IL-17; IL-22; Inflammation
The convergent distribution of the Human Immunodeficiency Virus (HIV) and helminth infections has led to the suggestion that infection with helminths exacerbates the HIV epidemic in developing countries. In South Africa, it is estimated that 57% of the population lives in poverty and carries the highest burden of both HIV and helmith infections, however, the disease interactions are under-researched.
We employed both coproscopy and Ascaris lumbricoides-specific serum IgE to increase diagnostic sensitivity and to distinguish between different helminth infection phenotypes and their effects on immune responses in HIV co-infected individuals. Coproscopy was done by formol ether and Kato Katz methods. HIV positive and negative adults were stratified according to the presence or absence of A. lumbricoides and/or Trichuris trichuria eggs with or without elevated Ascaris IgE. Lymphocyte subsets were phenotyped by flow cytometry. Viral loads, serum total IgE and eosinophils were also analysed. Lymphocyte activation markers (CCR5, HLA-DR, CD25, CD38 and CD71) were determined. Non parametric statistics were used to describe differences in the variables between the subgroups.
Helminth prevalence ranged between 40%-60%. Four distinct subgroups of were identified, and this included egg positive/high Ascaris-specific IgE (egg+IgEhi), egg positive/low IgE (egg+IgElo), egg negative/high IgE (egg-IgEhi) and egg negative/low IgE (egg-IgElo) individuals. The egg+IgEhi subgroup displayed lymphocytopenia, eosinophilia, (low CD4+ counts in HIV- group), high viral load (in HIV+ group), and an activated lymphocyte profile. High Ascaris IgE subgroups (egg+IgEhi and egg-IgEhi) had eosinophilia, highest viral loads, and lower CD4+ counts in the HIV- group). Egg excretion and low IgE (egg+IgElo) status demonstrated a modified Th2 immune profile with a relatively competent response to HIV.
People with both helminth egg excretion and high Ascaris-IgE levels had dysregulated immune cells, high viral loads with more immune activation. A modified Th2 helminth response in individuals with egg positive stools and low Ascaris IgE showed a better HIV related immune profile. Future research on helminth-HIV co-infection should include parasite-specific IgE measurements in addition to coproscopy to delineate the different response phenotypes. Helminth infection affects the immune response to HIV in some individuals with high IgE and egg excretion in stool.