Genetic variability and consequence of Mycobacterium tuberculosis lineage 3 in Kampala-Uganda

Limited data existed exclusively describing Mycobacterium tuberculosis lineage 3 (MTB-L3), sub-lineages, and clinical manifestations in Kampala, Uganda. This study sought to elucidate the circulating MTB-L3 sub-lineages and their corresponding clinical phenotypes.; A total of 141 M. tuberculosis isolates were identified as M. tuberculosis lineage 3 using Single nucleotide polymorphism (SNP) marker analysis method. To ascertain the sub-lineages/sub-strains within the M. tuberculosis lineage 3, the direct repeat (DR) loci for all the isolates was examined for sub-lineage specific signatures as described in the SITVIT2 database. The infecting sub-strains were matched with patients' clinical and demographic characteristics to identify any possible association.; The data showed 3 sub-lineages circulating with CAS 1 Delhi accounting for 55% (77/141), followed by CAS 1-Kili 16% (22/141) and CAS 2/CAS 8% (12/141). Remaining isolates 21% (30/141) were unclassifiable. To explore whether the sub-lineages differ in their ability to cause increased severe disease, we used extent of lung involvement as a proxy for severe disease. Multivariable analysis showed no association between M. tuberculosis lineage 3 sub-lineages with severe disease. The risk factors associated with severe disease include having a positive smear (OR = 9.384; CI 95% = 2.603-33.835), HIV (OR = 0.316; CI 95% = 0.114-0.876), lymphadenitis (OR = 0. 171; CI 95% = 0.034-0.856) and a BCG scar (OR = 0.295; CI 95% = 0.102-0.854).; In Kampala, Uganda, there are three sub-lineages of M. tuberculosis lineage 3 that cause disease of comparable severity with CAS-Dehli as the most prevalent. Having HIV, lymphadenitis, a BCG scar and a smear negative status is associated with reduced severe disease

Effect of maternal Schistosoma mansoni infection and praziquantel treatment during pregnancy on Schistosoma mansoni infection and immune responsiveness among offspring at age five years.

INTRODUCTION: Offspring of Schistosoma mansoni-infected women in schistosomiasis-endemic areas may be sensitised in-utero. This may influence their immune responsiveness to schistosome infection and schistosomiasis-associated morbidity. Effects of praziquantel treatment of S. mansoni during pregnancy on risk of S. mansoni infection among offspring, and on their immune responsiveness when they become exposed to S. mansoni, are unknown. Here we examined effects of praziquantel treatment of S. mansoni during pregnancy on prevalence of S. mansoni and immune responsiveness among offspring at age five years. METHODS: In a trial in Uganda (ISRCTN32849447, http://www.controlled-trials.com/ISRCTN32849447/elliott), offspring of women treated with praziquantel or placebo during pregnancy were examined for S. mansoni infection and for cytokine and antibody responses to SWA and SEA, as well as for T cell expression of FoxP3, at age five years. RESULTS: Of the 1343 children examined, 32 (2.4%) had S. mansoni infection at age five years based on a single stool sample. Infection prevalence did not differ between children of treated or untreated mothers. Cytokine (IFNγ, IL-5, IL-10 and IL-13) and antibody (IgG1, Ig4 and IgE) responses to SWA and SEA, and FoxP3 expression, were higher among infected than uninfected children. Praziquantel treatment of S. mansoni during pregnancy had no effect on immune responses, with the exception of IL-10 responses to SWA, which was higher in offspring of women that received praziquantel during pregnancy than those who did not. CONCLUSION: We found no evidence that maternal S. mansoni infection and its treatment during pregnancy influence prevalence and intensity of S. mansoni infection or effector immune response to S. mansoni infection among offspring at age five years, but the observed effects on IL-10 responses to SWA suggest that maternal S. mansoni and its treatment during pregnancy may affect immunoregulatory responsiveness in childhood schistosomiasis. This might have implications for pathogenesis of the disease

Development of a pHrodo-based assay for the assessment of in vitro and in vivo erythrophagocytosis during experimental trypanosomosis

Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis

pHrodo erythrophagocytosis assay using naïve PECs.

<p>A (left panel): CD11b versus F4/80 profile following gating on CD45+ cells allows identifying two distinct populations; (i) CD11b⁺F4/80⁺ cells which represent macrophages within the PEC population and (ii) cells which are negative or low for CD11b and F4/80 expression referred by as the Rest fraction. A (right panel): histogram showing the PE signal obtained following gating on CD11b⁺ F4/80⁺ cells when PECs are incubated alone (black line), with unlabeled RBCs (blue line), with pHrodo-labeled RBCs (orange line) or stimulated with LPS and incubated with pHrodo-labeled RBCs (green line). B: Histogram showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells incubated with unlabeled RBCs from cells incubated with pHrodo-labeled RBCs. PECS were unstimulated (white bars) or stimulated with 1μg/ml LPS (black bars). Results are presented +/- SEM and are representative of 4 independent experiments (for each point triplicates were used). (*: p-values ≤ 0.05) C: Microscopic pictures from PECs incubated with pHrodo-labeled RBCs.</p

Different myeloid sub-populations expressed as percentage within the CD45+ hematopoietic compartment or as absolute numbers within the organ.

<p>Left panels: Percentage of the different cell populations (neutrophils/PMN, monocytes, CD11b⁺F4/80⁺ myeloid cells and Rest fraction) within the CD45+ hematopoietic compartment obtained following gating of liver (A) and spleen (B) as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003561#pntd.0003561.s001" target="_blank">S1 Fig</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003561#pntd.0003561.s002" target="_blank">S2 Fig</a>, respectively, from non-infected (white bars) or <i>T</i>. <i>brucei</i> infected (day 6 p.i.) animals. Right panels: Absolute numbers of the different cell populations (neutrophils/PMN, monocytes, CD11b⁺F4/80⁺ myeloid cells and Rest fraction) in total liver and spleen of non-infected (white bars) and <i>T</i>. <i>brucei</i> infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.</p

Flow chart of the <i>in vitro</i> erythrophagocytosis assay protocol.

<p>Flow chart of the <i>in vitro</i> erythrophagocytosis assay protocol.</p

<i>In vivo</i> pHrodo erythrophagocytosis assay on liver and spleen cells during the acute stage of infection.

<p>A. Flow chart of the <i>in vivo</i> erythrophagocytosis assay protocol. To asses erythrophagocytosis in infected mice, donor mice should be sacrificed at day 5 post infection. Labeled blood is then transferred to 5 days-infected acceptor mice. At day 6 post infection the acceptor mice are sacrificed for organ isolation and flow cytometry analysis. As a control the same procedure is performed with naïve donor and acceptor mice. B. Erythrophagocytic potential of liver cell populations (neutrophils/PMN, monocytes, monocyte-derived macrophages, resident macrophages and rest fraction) from naïve (white bars) or <i>T</i>. <i>brucei</i> infected (day 6 p.i.). C. Erythrophagocytic potential of spleen cell populations (neutrophils/PMN, monocytes, CD11b⁺F4/80⁺ macrophages and rest fraction) from naïve (white bars) or <i>T</i>. <i>brucei</i> infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05 and if nothing is mentioned the differences were not significant.</p

pHrodo erythrophagocytosis assay using naïve spleen and liver cells.

<p>Histograms showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells incubated with unlabeled RBCs from cells incubated with pHrodo-labeled RBCs. Cells were either unstimulated (white bars) or stimulated with 1μg/ml LPS (black bars). Upper panel (A): Different liver cell populations (neutrophils, monocytes, CD11b⁺F4/80⁺ myeloid cells and Rest fraction) were identified as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003561#pntd.0003561.s001" target="_blank">S1 Fig</a>. Lower panel (B): Different spleen cell populations (neutrophils, monocytes, CD11b⁺F4/80⁺ myeloid cells and Rest fraction) were identified as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003561#pntd.0003561.s002" target="_blank">S2 Fig</a>. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.</p

A. Chart representing the lipid composition of mature RBCs from naïve and <i>T</i>. <i>brucei</i> infected mice.

<p>This result is a representative of three independent experiments, including three mice per time point. B. Osmotic fragility profile of RBCs following incubation of naïve (black line) or <i>T</i>. <i>brucei</i> infected (day 6 p.i.) (white line) with decreasing concentrations of NaCl, resulting in hemolysis of RBCs. The percentage of hemolysis was plotted against the concentration of NaCl in the medium and the NaCl concentrations corresponding with 50% hemolysis were determined. As positive control RBCs were exposed to 100% distilled H₂O and as negative control RBCs were exposed to 100% HBSS-solution. Results are representative of 3 independent experiments and expressed +/- SD. C. pHrodo erythrophagocytosis assay using RBCs from naïve and <i>T</i>. <i>brucei</i> infected mice co-cultured with PECs from non-infected (left panel) or infected (left panel) animals. Erythrophagocytosis by non-infected PECs of non-infected RBCs (white bars) or <i>T</i>. <i>brucei</i> infected (day 6 p.i.) (black bars). Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01.</p

Development of a Nanobody-based lateral flow assay to detect active Trypanosoma congolense infections

Animal African trypanosomosis (AAT), a disease affecting livestock, is caused by parasites of the Trypanosoma genus (mainly T. vivax and T. congolense). AAT is widespread in Sub-Saharan Africa, where it continues to impose a heavy socio-economic burden as it renders the development of sustainable livestock rearing very strenuous. Active case-finding and the identification of infected animals prior to initiation of drug treatment requires the availability of sensitive and specific diagnostic tests. In this paper, we describe the development of two heterologous sandwich assay formats (ELISA and LFA) for T. congolense detection through the use of Nanobodies (Nbs). The immunisation of an alpaca with a secretome mix from two T. congolense strains resulted in the identification of a Nb pair (Nb44/Nb42) that specifically targets the glycolytic enzyme pyruvate kinase. We demonstrate that the Nb44/Nb42 ELISA and LFA can be employed to detect parasitaemia in plasma samples from experimentally infected mice and cattle and, additionally, that they can serve as 'test-of-cure' tools. Altogether, the findings in this paper present the development and evaluation of the first Nb-based antigen detection LFA to identify active T. congolense infections