Anne Ma Dyrhol-Riise

Evolution of extensively drug-resistant Mycobacterium tuberculosis from a susceptible ancestor in a single patient

BackgroundMycobacterium tuberculosis is characterized by a low mutation rate and a lack of genetic recombination. Yet, the rise of extensively resistant strains paints a picture of a microbe with an impressive adaptive potential. Here we describe the first documented case of extensively drug-resistant tuberculosis evolved from a susceptible ancestor within a single patient.ResultsGenome sequences of nine serial M. tuberculosis isolates from the same patient uncovered a dramatic turnover of competing lineages driven by the emergence, and subsequent fixation or loss of single nucleotide polymorphisms. For most drugs, resistance arose through independent emergence of mutations in more than one clone, of which only one ultimately prevailed as the clone carrying it expanded, displacing the other clones in the process. The vast majority of mutations identified over 3.5 years were either involved in drug resistance or hitchhiking in the genetic background of these. Additionally, RNA-sequencing of isolates grown in the absence of drug challenge revealed that the efflux-associated iniBAC operon was up-regulated over time, whereas down-regulated genes include those involved in mycolic acid synthesis.ConclusionsWe observed both rapid acquisitions of resistance to antimicrobial compounds mediated by individual mutations as well as a gradual increase in fitness in the presence of antibiotics, likely driven by stable gene expression reprogramming. The rapid turnover of resistance mutations and hitchhiking neutral mutations has major implications for inferring tuberculosis transmission events in situations where drug resistance evolves within transmission chains.

Diagnosis and follow-up of treatment of latent tuberculosis; the utility of the QuantiFERON-TB Gold In-tube assay in outpatients from a tuberculosis low-endemic country

BackgroundInterferon-gamma (IFN-γ) Release Assays (IGRA) are more specific than the tuberculosis skin test (TST) in the diagnosis of latent tuberculosis (TB) infection (LTBI). We present the performance of the QuantiFERON®-TB Gold In-tube (QFT-TB) assay as diagnostic test and during follow-up of preventive TB therapy in outpatients from a TB low-endemic country.Methods481 persons with suspected TB infection were tested with QFT-TB. Thoracic X-ray and sputum samples were performed and a questionnaire concerning risk factors for TB was filled. Three months of isoniazid and rifampicin were given to patients with LTBI and QFT-TB tests were performed after three and 15 months.ResultsThe QFT-TB test was positive in 30.8% (148/481) of the total, in 66.9% (111/166) of persons with origin from a TB endemic country, in 71.4% (20/28) previously treated for TB and in 100% (15/15) of those diagnosed with active TB with no inconclusive results. The QFT-TB test was more frequently positive in those with TST ≥ 15 mm (47.5%) compared to TST 11-14 mm (21.3%) and TST 6-10 mm (10.5%), (p < 0.001). Origin from a TB endemic country (OR 6.82, 95% CI 1.73-26.82), recent stay in a TB endemic country (OR 1.32, 95% CI 1.09-1.59), duration of TB exposure (OR 1.59, 95% CI 1.14-2.22) and previous TB disease (OR 11.60, 95% CI 2.02-66.73) were all independently associated with a positive QFT-TB test. After preventive therapy, 35/40 (87.5%) and 22/26 (84.6%) were still QFT-TB positive after three and 15 months, respectively. IFN-γ responses were comparable at start (mean 6.13 IU/ml ± SD 3.99) and after three months (mean 5.65 IU/ml ± SD 3.66) and 15 months (mean 5.65 IU/ml ± SD 4.14), (p > 0.05).ConclusionOnly one third of those with suspected TB infection had a positive QFT-TB test. Recent immigration from TB endemic countries and long duration of exposure are risk factors for a positive QFT-TB test and these groups should be targeted through screening. Since most patients remained QFT-TB positive after therapy, the test should not be used to monitor the effect of preventive therapy. Prospective studies are needed in order to determine the usefulness of IGRA tests during therapy.

Adenosine Deaminase Activity Is a Sensitive Marker for the Diagnosis of Tuberculous Pleuritis in Patients with Very Low CD4 Counts

Background Adenosine Deaminase Activity (ADA) is a commonly used marker for the diagnosis of tuberculous pleural effusion. There has been concern about its usefulness in immunocompromised patients, especially HIV positive patients with very low CD4 counts. The objective of this study was to evaluate the sensitivity of ADA in pleural fluid in patients with low CD4 counts. Materials and Methods This was a retrospective case control study. Medical files of patients with tuberculous pleuritis and non-tuberculous pleuritis were reviewed. Clinical characteristics, CD4 cell counts in blood and biochemical markers in pleural fluid, including ADA were recorded. Results One ninety seven tuberculous pleuritis and 40 non- tuberculous pleuritis patients were evaluated. Using the cut-off value of 30 U/L, the overall sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of ADA was 94%, 95%, 19, and 0.06 respectively. The mean CD4 cell counts among TB pleuritis patients was 29 and 153 cells/microL in patients with CD4 <50 cells/microL and >50 cells/microL, (p<0.05) respectively. The corresponding mean ADA values for these patients were 76 U/L and 72 U/L respectively (p>0.5). There was no correlation between ADA values and CD4 cell counts (r = −0.120, p = 0.369). Conclusion ADA analysis is a sensitive marker of tuberculous pleuritis even in HIV patients with very low CD4 counts in a high TB endemic region. The ADA assay is inexpensive, rapid, and simple to perform and is of great value for the immediate diagnosis of tuberculous pleuritis while waiting for culture result and this has a positive impact on patient outcome.

Activation of CD8 T cells normalizes and correlates with the level of infectious provirus in tonsils during highly active antiretroviral therapy in early HIV-1 infection.

OBJECTIVES To study the effects of antiretroviral therapy on T cell activation in blood and tonsils from HIV-1 infected individuals in relation to CD4 cell count, plasma viremia, and infectious HIV-1 provirus. DESIGN A 48-week study of viral load and T cell subsets in blood and tonsils from 12 HIV-1-positive individuals with a mean CD4 cell number of 400 x 10(6) cells/l treated with a combination of zidovudine, lamivudine, and indinavir. METHODS Tonsil biopsies and blood samples were collected at regular intervals. Lymphocytes were phenotyped and quantified by three-color flow cytometry; infectious provirus was quantified by a limiting dilution assay. HIV-1-negative individuals were included as controls. RESULTS The fraction of tonsillar CD8 T cells expressing CD69, CD38, or HLA-DR in the patients with suppressed virus replication declined to levels comparable with that in controls by 48 weeks and showed a strong positive correlation with tonsillar infectious provirus and plasma viremia. The level of CD4 T cell activation was within normal range in tonsils throughout the study. The fraction of HLA-DR+ cells within CD4 and CD8 T cells in blood declined rapidly in parallel with plasma viremia but remained slightly higher compared with that in uninfected individuals. CONCLUSION Antiretroviral therapy normalizes tonsillar CD8 T cell activation in HIV-1-positive individuals in parallel with suppression of viral replication, indicating reduced CD8 cell turnover. Normal tonsillar CD4 T cell activation suggests limited CD4 cell turnover in early HIV infection. Activated CD8 T cells in lymphoid tissue is superior to that in blood as an immunological marker for the virological response to antiretroviral therapy.

Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area.

The aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis.

T regulatory cells and immune activation in Mycobacterium tuberculosis infection and the effect of preventive therapy.

Mycobacterium tuberculosis (TB) often causes persistent infection and many immune cell subsets and regulatory mechanisms may operate throughout the various stages of infection. We have studied dendritic cell (DC) subsets, regulatory T cells (Treg) and the expression of activation and apoptosis markers on CD4+ and CD8+ T cells in blood from patients with active TB (n = 20), subjects with positive QuantiFERON‐TB GOLD (QFT) test (LTBI, latent TB infection) (n = 20) before and after 3 months of preventive anti‐tuberculous therapy and from QFT‐negative controls (n = 28). The frequency of CD4+CD25+CD127− Treg was highest in the group with active TB (P = 0.001), but also increased in the LTBI group (P = 0.006) compared to controls. The highest level of activated T cells, defined as CD38+HLA‐DR+ cells, was found in the active TB group, for the CD4+ T cell subset positively correlated to the level of CD25+CD127− Treg (P < 0.001, r = 0.4268). After 3 months of preventive therapy, there was an increase in the fraction of foxp3+ Treg, but no differences in markers of activation or apoptosis. In conclusion, there seems to be an increased level of immune activation and Treg in both latent and active TB infection that is only modestly influenced by preventive therapy.

IP-10 differentiates between active and latent tuberculosis irrespective of HIV status and declines during therapy

OBJECTIVES Biomarkers for diagnosis and therapy efficacy in tuberculosis (TB) are requested. We have studied biomarkers that may differentiate between active and latent TB infection (LTBI), the influence of HIV infection and changes during anti-TB chemotherapy. METHODS Thirty-eight plasma cytokines, assessed by multiplex and enzyme immunoassays, were analyzed in patients with active TB before and during 24 weeks of anti-TB chemotherapy (n = 65), from individuals with LTBI (n = 34) and from QuantiFERON-TB (QFT) negative controls (n = 65). The study participants were grouped according to HIV status. RESULTS Plasma levels of the CXC chemokine IP-10 and soluble TNF receptor type 2 (sTNFr2) significantly differentiated active TB from the LTBI group, irrespective of HIV status. In the HIV-infected group the sensitivity and specificity was 100% for IP-10 with a cut-off of 2547 pg/mL. Plasma IP-10 declined gradually during anti-TB chemotherapy (12-24 weeks, p = 0.002) to a level comparable to LTBI and QFT negative control groups. sTNFr2 fluctuated throughout therapy, but was decreased after 12-24 weeks (p = 0.006). CONCLUSIONS IP-10 distinguished with high accuracy active TB from LTBI irrespective of HIV infection and declined during anti-TB chemotherapy. Plasma IP-10 may serve as a diagnostic biomarker to differentiate between the stages of TB infection and for monitoring therapy efficacy.

Reduced Levels of D-dimer and Changes in Gut Microbiota Composition After Probiotic Intervention in HIV-Infected Individuals on Stable ART

Background:Microbial translocation and chronic inflammation may contribute to non-AIDS morbidity in patients with HIV. This study assessed the impact of probiotic intervention on microbial translocation and inflammation in patients on antiretroviral therapy with viral suppression and subnormal CD4 count. Methods:Thirty-two patients receiving antiretroviral therapy (CD4 <500 cells/&mgr;L) were randomized in a double-blind fashion to multistrain daily probiotics (n = 15), placebo (n = 9), or controls (n = 8) for 8 weeks. Soluble inflammation markers, D-dimer, lipopolysaccharide (LPS), sCD14, T-cell activation, tryptophan metabolites, and gut microbiota composition were analyzed at baseline and end of study. Nonparametric statistics were applied. Results:Twenty-four participants completed the study and were included in as-treated analyses. In patients receiving probiotics, there was a significant reduction in D-dimer levels (median change 33%, P = 0.03) and a tendency to reduced levels of C-reactive protein (CRP) (P = 0.05) and interleukin (IL)-6 (P = 0.06). The changes in CRP and IL-6 were highly correlated (r = 0.95, P < 0.01), whereas changes in D-dimer did not correlate with changes in CRP or IL-6. Increases in Bifidobacteria (P = 0.04) and Lactobacilli (P = 0.06) were observed in the probiotic group, whereas the relative abundance of Bacteroides decreased (P ⩽ 0.01). No significant changes were seen in markers of microbial translocation or T-cell activation. However, the expansion of Bifidobacteria correlated negatively with differences in LPS (r = −0.77, P = 0.01), whereas the reduction in Bacteroides correlated positively with changes in LPS during the study period (r = 0.72, P = 0.02). Conclusions:Probiotic intervention seemed to reduce markers of coagulation and inflammation without overt changes in microbial translocation. These findings warrant further studies in larger cohorts with long-term follow-up.

Early dynamics of T helper cell cytokines and T regulatory cells in response to treatment of active Mycobacterium tuberculosis infection

Biomarkers that can identify tuberculosis (TB) disease and serve as markers for efficient therapy are requested. We have studied T cell cytokine production [interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐α] and degranulation (CD107a) as well as subsets of CD4+ T regulatory cells (Tregs) after in‐vitro Mycobacterium tuberculosis (Mtb) antigen stimulation [early secretory antigenic target (ESAT)‐6, culture filtrate protein (CFP)‐10, antigen 85 (Ag85)] in 32 patients with active tuberculosis (TB) disease throughout 24 weeks of effective TB treatment. A significant decline in the fraction of Mtb‐specific total IFN‐γ and single IFN‐γ‐producing T cells was already observed after 2 weeks of treatment, whereas the pool of single IL‐2+ cells increased over time for both CD4+ and CD8+ T cells. The Treg subsets CD25highCD127low, CD25highCD147++ and CD25highCD127lowCD161+ expanded significantly after Mtb antigen stimulation in vitro at all time‐points, whereas the CD25highCD127lowCD39+ Tregs remained unchanged. The fraction of CD25highCD127low Tregs increased after 8 weeks of treatment. Thus, we revealed an opposing shift of Tregs and intracellular cytokine production during treatment. This may indicate that functional signatures of the CD4+ and CD8+ T cells can serve as immunological correlates of early curative host responses. Whether such signatures can be used as biomarkers in monitoring and follow‐up of TB treatment needs to be explored further.

Changes in immunoglobulin isotypes and immunoglobulin G (IgG) subclasses during highly active antiretroviral therapy: Anti-p24 IgG1 closely parallels the biphasic decline in plasma viremia

The effects of highly active antiretroviral therapy (HAART) on immunoglobulin isotypes and immunoglobulin G (IgG) subclasses were studied in 12 patients in early stages of HIV-1 infection. Blood samples were obtained at enrollment and 2, 4, 8, 12, 24, 48, and 120 weeks after initiation of HAART. Immunoglobulin concentrations were determined by nephelometry, and anti-p24–specific IgG and IgG1 levels were determined by an enzyme immunoassay. Overall time changes were analyzed in analysis of variance models. IgG and IgG1 levels showed a marked overall decline, whereas other immunoglobulin isotypes and IgG subclasses did not change significantly. Anti-p24–specific IgG1 levels decreased considerably and significantly more in virus isolation–negative patients than in virus isolation–positive patients, as defined according to the ability to isolate HIV-1 from their CD4+ T cells after initiation of therapy. Anti-p24 IgG levels showed a similar but overall weaker decline in the two groups. However, the anti-p24 IgG1 level followed the biphasic decline in plasma viremia more closely than the anti-p24 IgG level, with an initial sharp decline that leveled off with time. These findings suggest that the main reduction in immunoglobulin levels is caused by reduced HIV-1–specific antigen stimulation rather than a general reduction in immune activation. Using anti-p24 IgG1 as a parameter of response to the effect of HAART merits further investigation.