Confocal Laser Scanning Microscopy, a New In Vivo Diagnostic Tool for Schistosomiasis

BACKGROUND: The gold standard for the diagnosis of schistosomiasis is the detection of the parasite's characteristic eggs in urine, stool, or rectal and bladder biopsy specimens. Direct detection of eggs is difficult and not always possible in patients with low egg-shedding rates. Confocal laser scanning microscopy (CLSM) permits non-invasive cell imaging in vivo and is an established way of obtaining high-resolution images and 3-dimensional reconstructions. Recently, CLSM was shown to be a suitable method to visualize Schistosoma mansoni eggs within the mucosa of dissected mouse gut. In this case, we evaluated the suitability of CLSM to detect eggs of Schistosoma haematobium in a patient with urinary schistosomiasis and low egg-shedding rates. METHODOLOGY/PRINCIPAL FINDINGS: The confocal laser scanning microscope used in this study was based on a scanning laser system for imaging the retina of a living eye, the Heidelberg Retina Tomograph II, in combination with a lens system (image modality). Standard light cystoscopy was performed using a rigid cystoscope under general anaesthesia. The CLSM endoscope was then passed through the working channel of the rigid cystoscope. The mucosal tissue of the bladder was scanned using CLSM. Schistoma haematobium eggs appeared as bright structures, with the characteristic egg shape and typical terminal spine. CONCLUSION/SIGNIFICANCE: We were able to detect schistosomal eggs in the urothelium of a patient with urinary schistosomiasis. Thus, CLSM may be a suitable tool for the diagnosis of schistosomiasis in humans, especially in cases where standard diagnostic tools are not suitable

Confocal Laser Scanning Microscopy for Detection of Schistosoma mansoni Eggs in the Gut of Mice

Background: The gold standard for diagnosing Schistosoma mansoni infections is the detection of eggs from stool or biopsy specimens. The viability of collected eggs can be tested by the miracidium hatching procedure. Direct detection methods are often limited in patients with light or early infections, whereas serological tests and PCR methods fail to differentiate between an inactive and persistent infection and between schistosomal species. Recently, confocal laser scanning microscopy (CLSM) has been introduced as a diagnostic tool in several fields of medicine. In this study we evaluated CLSM for the detection of viable eggs of S. mansoni directly within the gut of infected mice. Methodology/Principal Findings: The confocal laser scanning microscope used in this study is based on the Heidelberg Retina Tomograph II scanning laser system in combination with the Rostock Cornea Module (image modality 1) or a rigid endoscope (image modality 2). Colon sections of five infected mice were examined with image modalities 1 and 2 for schistosomal eggs. Afterwards a biopsy specimen was taken from each colon section and examined by bright-field microscopy. Visualised eggs were counted and classified in terms of viability status. Conclusions/Significance: We were able to show that CLSM visualises eggs directly within the gut and permits discrimination of schistosomal species and determination of egg viability. Thus, CLSM may be a suitable non-invasive too

Suppression of TGF-β pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro

Stahnke T, Kowtharapu BS, Stachs O, et al. Suppression of TGF-β pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro. PLOS ONE. 2017;12(2): e0172592

Confocal laser scanning microscopy of the urinary bladder.

<p><i>In vivo</i> confocal laser scanning microscopy of the bladder showing eggs of <i>Schistosoma haematobium</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034869#pone-0034869-g001" target="_blank">figure 1a and 1b</a>) with their typical terminal spine (arrow) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034869#pone-0034869-g001" target="_blank">figure 1b</a>).</p

Relative proliferation of hTFs and hOFs in response to TGF-β1 and PFD <i>in vitro</i>.

<p>Fibroblasts were treated with TGF-β1 [10 ng/ml], PFD [10⁻³ mol/l] or the combination of TGF-β1 [10 ng/ml] and PFD [10⁻³ mol/l] for 48 h under serum-free (A and C) and serum (10% FCS) conditions (B and D) as indicated. NC (proliferation rate of untreated cells) was set to 100%. Data are presented as mean ± SD. The results represent the means of three independent experiments. Level of significances: *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.</p

Antibodies used in this study.

<p>Antibodies used in this study.</p

Human fibroblast subpopulations <i>in vitro</i>.

<p>Primary fibroblast subpopulations from Tenon’s capsule (hTFs) and fibroblasts from orbital fat (hOFs) were cultured for 1, 3 and 7 days, respectively. div = days <i>in vitro</i>. Bar represents 25 μm.</p

RT-PCR analysis of stimulated (TGF-β1) and suppressed (PFD) primary hOFs.

<p>Cultures of hOFs were treated with TGF-β1 [10 ng/ml], PFD [10⁻³ mol/l] or the combination of both, TGF-β1 and PFD for 48 h under serum-free culture conditions. NC (expression level of respective gene in untreated cells, dotted line) was set to 1. Data are presented as mean ± SD. The results represent the means of four independent experiments. Level of significances: *p≤0.05; **p≤0.01; ***p≤0.001. Overall, no statistically significant differences between TGF-β1 and TGF-β1+PFD groups could be observed.</p

Immunocytochemical characterization of TGF-β1 induced fibronectin and α-SMA expression in primary hOFs <i>in vitro</i>.

<p>After PFA fixation, cells were incubated with primary antibodies directed against fibronectin and α-SMA. Nuclei were stained by DAPI included in the mounting medium. PFD decreased fibronectin and α-SMA expression. Experiments were carried out four times with similar results. Bar represents 50 μm.</p

Immunocytochemical characterization of TGF-β1 induced fibronectin and α-SMA expression in primary hTFs <i>in vitro</i>.

<p>After PFA fixation, cells were incubated with primary antibodies directed against fibronectin and α-SMA. Nuclei were stained by DAPI included in the mounting medium. PFD decreased fibronectin and α-SMA expression. Experiments were carried out four times with similar results. Bar represents 50 μm.</p