Direct detection and quantification of MABSC by CFU, DNA-strip, and rpoB-qPCR of non-cultured CF sputum samples.

<p>(A) Direct <i>ex vivo</i> PCR strip results of sputum from MABSC-positive CF patients (i.e. 034, 023, 022, 029) and CF patient 002 (previous MABSC infection but unclear infection status). A band pattern at positions 3, 5, 6, and 10 indicates MABSC (the same pattern but absence of a band at position 3 and 6 indicates either <i>M. abscessus</i> or <i>M. chelonae</i>). The CC band is a conjugate control, the UC band indicates bacterial DNA with a high GC-content, the GC band indicates DNA of the genus <i>mycobacterium</i>. (B) CFU (colony forming units), <i>rpoB</i>-qPCR (both at the y-axis), and PCR strip test results (positive tests indicated as +, negative as-) for serial dilution (10-fold) of MABSC in sputum. Dark grey bars indicate CFU numbers. Bright grey bars indicate normalized <i>rpoB</i> copies. Mean values of triplicate samples and standard deviations are given. One representative experiment of two is shown. (C) <i>RpoB</i>-qPCR results of sputum from MABSC-positive CF patients (i.e. 034, 023, 022, 029) and CF patient 002 are depicted. Calculated MABSC sputum concentrations (adjusted for 50-fold lower sensitivity of rpo-qPCR as compared to MABSC culture) are given. (D) Time course analyses of <i>rpoB</i>-qPCR and mycobacterial sputum culture from CF patient CF034. Circles depict <i>rpoB</i>-qPCR values and arrows indicate time points of MABSC sputum culture. nd (not detectable) indicates <i>rpoB</i>-qPCR runs where MABSC <i>rpoB</i> was not detected. The dotted line indicates the detection limit of qPCR.</p

Primers and probe used for qPCR

<p>Primers and probe used for qPCR</p

Rapid Detection and Immune Characterization of <i>Mycobacterium abscessus</i> Infection in Cystic Fibrosis Patients

<div><p>Cystic fibrosis patients are highly susceptible to infections with non-tuberculous mycobacteria. Especially <i>Mycobacterium abscessus</i> infections are common but reliable diagnosis is hampered by non-specific clinical symptoms and insensitive mycobacterial culture. In the present study we established novel methods for rapid detection and immune characterization of <i>Mycobacterium abscessus</i> infection in cystic fibrosis patients. We performed <i>Mycobacterium abscessus</i> specific DNA-strip- and quantitative PCR-based analyses of non-cultured sputum samples to detect and characterize <i>Mycobacterium abscessus</i> infections. Concomitantly <i>in vitro</i> T-cell reactivation with purified protein derivatives (PPDs) from different mycobacterial species was used to determine <i>Mycobacterium abscessus</i> specific T-cell cytokine expression of infected cystic fibrosis patients. Four of 35 cystic fibrosis patients (11.4%) were <i>Mycobacterium abscessus</i> culture positive and showed concordant DNA-strip-test results. Quantitative PCR revealed marked differences of mycobacterial burden between cystic fibrosis patients and during disease course. Tandem-repeat analysis classified distinct <i>Mycobacterium abscessus</i> strains of infected cystic fibrosis patients and excluded patient-to-patient transmission. <i>Mycobacterium abscessus</i> specific T-cells were detected in the blood of cystic fibrosis patients with confirmed chronic infection and a subgroup of patients without evidence of <i>Mycobacterium abscessus</i> infection. Comparison of cytokine expression and phenotypic markers revealed increased proportions of CD40L positive T-cells that lack Interleukin-2 expression as a marker for chronic <i>Mycobacterium abscessus</i> infections in cystic fibrosis patients. Direct sputum examination enabled rapid diagnosis and quantification of <i>Mycobacterium abscessus</i> in cystic fibrosis patients. T-cell <i>in vitro</i> reactivation and cytokine expression analyses may contribute to diagnosis of chronic <i>Mycobacterium abscessus</i> infection.</p></div

Study results.

<p>* One CF patient (CF002) had a history of previous MABSC infection but an unclear infection status at study onset due to persistent culture contamination. For sputum PCR analyses this patient was classified as MABSC negative (confirmed by our assays). n: number of patients; na: not applicable; pos: positive; neg: negative.</p><p>Study results.</p

Clinical characteristics of CF patients

<p>n: number of patients; f/m: number of females/males; FEV: forced expiratory volume; BMI: body mass index</p><p>Clinical characteristics of CF patients</p

Gating strategy of cytokine-expressing T-cell proportions.

<p>Density plots (left graphs) and dot plots (right graphs) depict flow cytometry analyses of data of whole blood <i>in vitro</i> restimulation. Results of stimulation with different mycobacterial antigens, i.e. abscessin of <i>M. abscessus</i> (MABSC), sensitin of <i>M. avium</i> (MAC), and tuberculin of <i>M. tuberculosis</i> (MTB), the non-clonal T-cell activator SEB (Staphylococcus enterotoxin B), and without stimulation (w/o) are shown. Grey block-arrows indicate the sequence of analyses steps. After excluding cell doublets by comparing FSC-A (forward scatter area) and FSC-H (forward scatter height) parameters (upper left density plot), lymphocytes were selected on the basis of cellular size (FSC-A) and granularity (SSC-A; side scatter area). Viable CD4⁺ memory T-cells (CD45RA negative) were selected for cytokine analysis. Proportions of TNFα/IL-2, CD40L/IL-2, and IFNɣ/IL-2 double positive T-cells were determined for each stimuli (right graphs). A representative analysis of a MABSC infected CF patient is shown.</p

Cytokine expression of abscessin specific T-cells.

<p>MABSC induced cytokine-expressing T-cell proportions of CF patients (n = 35). (A) Abscessin specific T-cells that produce IL-2 together with either CD40L (upper graph), or IFNɣ (middle graph), or TNFα (lower graph) are shown. Bars indicate mean and standard deviation of measured duplicates for each individual patient (ID label on the x-axis). Green color indicates MABSC confirmed cases. Blue color indicates CF patients without MABSC infection (current and past) but a positive T-cell response against MABSC, defined by at least two of three cytokine combinations values [mean proportions] above 0.03% [3-times the assumed flow cytometry detection limit] (B) Comparisons of T-cell cytokine responses induced by abscessin, tuberculin, and sensitin. Ratios (abscessin / tuberculin or abscessin / sensitin) for CD40L/IL-2 positive T-cells are shown for confirmed MABSC infected CF patients (green triangles) and non-confirmed MABSC responders (blue circles). Each symbol represents ratios calculated of mean proportions for an individual donor. The dotted lines indicate equal T-cell responses (ratio = 1) (C) Relative T-cell responses against abscessin, tuberculin, and sensitin are depicted in a ternary plot. The sum of T-cell proportions for each individual donor was set to 1. Relative values of specific T-cells for each PPD are indicated in a ternary plot. MABSC infected CF patients are depicted as green triangles and non-confirmed MABSC responders as blue circles. The MABSC non-confirmed CF patient CF030 is highlighted who has 50% abscessin, 40% sensitin, and 10% tuberculin specific T-cells.</p

Characterization of MABSC strains from CF patients by VNTR (Variable Number Tandem Repeat) analysis.

<p>VNTR-PCR results of MABSC-confirmed CF patients are depicted. Agarose gel electrophoresis for six tandem repeat regions are shown. Differences in the band pattern indicate MABSC strain specific differences.</p

Proportional and absolute differences of LD-BCG infected MDM and comparison with Colony Forming Units (CFU) and fluorescence microscopy.

<p>Rifampicin treatment of MDM infected with LD-BCG at different indicated concentrations is shown. (A) Flow cytometry (FACS) analysis of MDM proportions infected with live (grey) or dead (open) LD-BCG are shown as stacked boxes. (B) Comparison of absolute MDM numbers infected with LD-BCG measured by bead corrected flow cytometry. Bead-based standardization of measured sample volume is shown in the upper graphs. Absolute numbers of all infected MDM (lower left graph) as well as MDM containing live and dead LD-BCG (lower right graph) at different rifampicin concentrations are shown. Median with range of triplicates are depicted. (C) Comparison of live LD-BCG infected MDM numbers measured by FACS and mycobacterial culture (CFU). Circles indicate FACS values adjusted for multiple infections as determined by fluorescence microscopy. Median values with range of triplicates are depicted. (D) Fluorescence microscopy analyses of MDMs infected with LD-BCG with or w/o ATC or non-infected MDM are shown. Blue color indicates MDM nuclei; red color indicates mCherry expressing LD-BCG; green color indicates GFP-expressing live LD-BCG. A representative experiment of three is shown.</p

Cytotoxic effects of total effector T cells and T-cell subpopulations against LD-BCG infected MDM and markers of induced cell death on MDM.

<p>Combined analyses of autologues E/M samples from healthy donors (n = 8) for (A), (n = 6) for (B), and (n = 5) for (C) are shown. Total effector T cells (A) or enriched CD4⁺/CD8⁺ T-cell subpopulations (B, C) were applied. (A, B) Cytotoxicity of differentially stimulated effector T cells against MDM infected with LD-BCG was assessed by flow cytometry. Different E/M ratios were applied (x-axes) and absolute numbers of MDM infected with live LD-BCG are indicated (y-axes). Mean and standard deviations are shown. (C) Apoptosis marker expression on MDM with or w/o LD-BCG infection after coculture with differentially stimulated CD4⁺ or CD8⁺ effector T cells. E:M ratios of 1:3 are shown. The proportions of non-apoptotic (open), early apoptotic (bright grey), and late apoptotic (dark grey) MDM are depicted as pie charts. Non-infected MDM (left panel) as well as MDM infected with live (middle) and dead (left) LD-BCG are shown. Median values of five independent experiments are depicted. The Mann-Whitney U-test were applied. Asterisks indicate significant differences (***: p<0.001; **: p<0.01; *: p<0.05).</p