Binding and uptake of B3501 and B-4131 with human brain endothelial cell line, hCMEC/D3.

<p>HCMEC/D3 cells were exposed to <i>C. neoformans</i> serotype D wild type B3501 and its isogenic acapsular mutant B4131 for 2 hr and 4 hr at 37°C. Microscopic and live CFU counts were performed to determine the association and survival of cryptococci. (A) Association efficiency of B3501 and B4131 cryptococci with hCMEC/D3 cells. Like the H99-cap59 pair, live CFU counts showed a time dependent decrease in the number of associated cryptococci by 4 hr of incubation, ★ = P<0.01, ★★ = P<0.01 for both B3501 and B4131. There was no difference in association by encapsulated B3501 and acapsular B4131, P-value = >0.05 at 2 hr of incubation. However, B4131 was significantly more associated, P = 0.02 (Microscopy) and <0.01 (CFU) by 4 hr of incubation. (B) Internalization of encapsulated B3501 and its acapsular mutant derivative B4131 by hCMEC/D3 cells determined by fluorescence microscopy (Mic). The number of phagocytosed cryptococci increased significantly in a time dependent manner, P = 0.01 for both strains. However, there was no difference in internalization of B3501 and B4131, P = 0.5 and 0.2 at 2 hr and 4 hr respectively. Error bars are standard error of the mean, n = 6 repeats.</p

Binding and uptake of H99 and cap59 with human brain endothelial cell line, hCMEC/D3.

<p>Like bEnd3 cells, hCMEC/D3 cells were exposed to the <i>C. neoformans</i> serotype A isogenic strains, wild type H99 and acapsular derivative cap59 for 2 hr and 4 hr at 37°C. Microscopic (Mic.) and live CFU counts were performed to determine the association and survival of cryptococci. (A) Association efficiency of H99 and cap59 cryptococci with hCMEC/D3 cells. As opposed to microscopic counts, live CFU counts showed a time dependent decrease in the number of associated cryptococci by 4 hr of incubation, ★ = P<0.05 and ★★ = P<0.01 for H99 and cap59 respectively, suggesting a drop in viability of the hCMEC/D3 associated H99 and cap59 cryptococci. There was no difference in association of encapsulated H99 and acapsular cap59, P = 0.7 and 0.6 at 2 hr and 4 hr respectively. (B) Internalization of encapsulated H99 and acapsular cap59 cryptococci by human brain endothelial cell line, hCMEC/D3 cells. The internalized cryptococci and extracellular adherent were determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035455#pone-0035455-g001" target="_blank">Figure 1</a>. As with association, the number of phagocytosed cryptococci increased significantly in a time dependent manner, P = 0.01 and <0.01 for H99 and cap59 respectively. However, there was no difference in internalization of H99 and acapsular cap59 cryptococci, P = 0.5 and 0.8 at 2 hr and 4 hr respectively. Error bars are standard error of the mean, n = 6 repeats.</p

Binding and uptake of H99 and cap59 to the murine brain endothelial cell line, bEnd3.

<p>BEnd3 cells were exposed to wild type H99 and its acapsular derivative cap59 for 2 hr and 4 hr at 37°C. (A) The rate of association (bound and internalized cryptococci) was determined both by microscopy and live CFU counts. The rate of association increased significantly in a time dependent manner, (P<0.01 for both strains). However, there was no difference in association of encapsulated H99 or acapsular cap59 cells (P = 0.1 and 0.5 at 2 hr and 4 hr respectively). (B) Internalization of the two strains, H99 and cap59 by bEnd3 cells as determined by fluorescence microscopy. Internalized cryptococci (pre-stained with FITC) were distinguished from extracellular adherent ones by counter labelling with calcofluor white after infection. Intracellular cryptococci retained the FITC green signal while extracellular cells acquired the blue signal from calcofluor. The number of phagocytosed cryptococci increased significantly in a time dependent manner, (P<0.01 for both strains). However, there was no difference in internalization of encapsulated H99 and acapsular cap59, (P = 0.5 and 0.8 at 2 hr and 4 hr respectively). Error bars are standard error of the mean, n = 5 repeats.</p

Effect of viability on binding and phagocytosis of H99 and cap59 strains by BMEC.

<p>Heating for 15 min at 65°C prior to infection killed H99 and cap59 cryptococci. Brain microvascular cells (BMEC), bEnd3 and or hCMEC/D3 cells were exposed in parallel to live (LV) and heat-killed (HK) cryptococci for 2 hr and 4 hr at 37°C and the number of bound and internalized cryptococci was determined by fluorescence microscopy. (A and C) Association efficiency of live and heat-killed H99 or cap59 cryptococci by bEnd3 and hCMEC/D3 cell respectively. There was no difference in association of viable H99 and cap59 cryptococci with hCMEC/D3 cells at either time point, P>0.05. However, viable cryptococci were more associated than non-viable ones by 4 hr of incubation in bEnd3 cells, P<0.01 for both H99 and cap59 respectively. (B and D) Internalization efficiency of LV and HK H99 and cap59 cryptococci by bEnd3 and hCMEC/D3 cells. Both live and heat-killed cryptococci showed a time dependent increase in phagocytosis by both bEnd3 and hCMEC/D3 cells, however the rate of phagocytosis did not vary between the viable and non-viable cryptococci in both cell-lines, P>0.05 at both 2 hr and 4 hr of incubation. Error bars are standard error of the mean, n = 5 repeats.</p

Effect of opsonisation on cryptococcal binding and uptake by bEnd3 cells.

<p>Live (LV) and heat-killed (HK) H99 cryptococci were opsonised with mouse derived anti-capsule IgG antibody, 18B7 and the rate of adherence and internalization was determined by fluorescence microscopy. (A) Association of opsonised (op) and non-opsonised (Nop) cryptococci with bEnd3 cells. Rate of association of opsonised and non-opsonised H99LV cryptococci was similar, P = 0.8 and 0.4 at 2 hr and 4 hr respectively. Similarly, there was no difference between opsonised and non-opsonised heat-killed H99 cryptococci, P = 0.9 and 0.5 at 2 hr and 4 hr respectively. (B) Rate of internalization of opsonised and non-opsonised cryptococci internalized by bEnd3 cells. The number of internalized H99LV cryptococci was similar, regardless of opsonisation status (P = 0.9 at both 2 hr and 4 hr). Similarly, there was no difference between opsonised and non-opsonised heat-killed H99 cryptococci, P = 0.5 and 0.4 at 2 hr and 4 hr respectively. Error bars are standard error of the mean, n = 5 repeats.</p

Data_Sheet_1_Systematic assessment of clinical and bacteriological markers for tuberculosis reveals discordance and inaccuracy of symptom-based diagnosis for treatment response monitoring.docx

BackgroundClinical symptoms are the benchmark of tuberculosis (TB) diagnosis and monitoring of treatment response but are not clear how they relate to TB bacteriology, particularly the novel tuberculosis-molecular bacterial load assay (TB-MBLA).MethodsPresumptive cases were bacteriologically confirmed for TB and assessed for symptoms and bacteriological resolution using smear microscopy (SM), culture, and TB-MBLA over 6-month treatment course. Kaplan–Meier and Kappa statistics were used to test the relationship between symptoms and bacteriological positivity.ResultsA cohort of 46 bacteriologically confirmed TB cases were analyzed for treatment response over a 6-month treatment course. Pre-treatment symptoms and bacteriological positivity concurred in over 70% of the cases. This agreement was lost in over 50% of cases whose chest pain, night sweat, and loss of appetite had resolved by week 2 of treatment. Cough resolved at a 3.2% rate weekly and was 0.3% slower than the combined bacteriological (average of MGIT and TB-MBLA positivity) resolution rate, 3.5% per week. A decrease in TB-MBLA positivity reflected a fall in bacillary load, 5.7 ± 1.3- at baseline to 0.30 ± 1.0- log10 eCFU/ml at month 6, and closer to cough resolution than other bacteriological measures, accounting for the only one bacteriologically positive case out of seven still coughing at month 6. Low baseline bacillary load patients were more likely to be bacteriologically negative, HR 5.6, p = 0.003 and HR 3.2, p = 0.014 by months 2 and 6 of treatment, respectively.ConclusionThe probability of clinical symptoms reflecting bacteriological positivity weakens as the patient progresses on anti-TB therapy, making the symptom-based diagnosis a less reliable marker of treatment response.</p

Median <i>in vitro</i> phenotyping values ordered by molecular type, VNI Subtype and high frequency MLST.

<p>Kruskal Wallis analysis performed on groups, and p values shown. Overall median is plotted in red. (A) Survival in <i>ex vivo</i> CSF, (B) Laccase Activity normalised to H99 reference strain, (C) <i>In vitro</i> phagocytosis of isolates by J774 cells (per 1 μl lysate).</p

<i>In vitro</i> phenotyping results ordered by molecular type, VNI subtype, and high frequency MLST type.

<p>* CSF survival was not performed for one of the eight VNB isolates.</p><p><i>In vitro</i> phenotyping results ordered by molecular type, VNI subtype, and high frequency MLST type.</p

Phylogenetic and Bayesian analysis of concatenated nucleotide sequences.

<p>(A) SplitsTree neighbour network shows diverse VNII and VNB, and clonal VNI clades. (B) Analysis of VNI clustering using StructureHARVESTER and ΔK shows optimal number of K clusters is 3. (C) Use of STRUCTURE allows VNI sequences to be subdivided into 3 distinct populations: VNI(a), VNI(b), VNI(c).</p

Assignment of MLST alleles, phylogeny and distribution of clinical isolates.

<p>Unrooted, ordered maximum likelihood tree (RAxML, GTR gamma, partitioned by loci) using concatenated nucleotide sequences from 7 loci and one representative sequence for each MLST type. Bootstrap values are shown for branches with greater than 60% agreement between replicates (1000 replicates). Also showing allele typing and MLST assignment for each sequence type, molecular type assignment according to phylogeny, and number of isolates belonging to each ST type.</p