Evaluation of Polymerase Chain Reaction (PCR) with Slit Skin Smear Examination (SSS) to Confirm Clinical Diagnosis of Leprosy in Eastern Nepal

<div><p>Background</p><p>Detection of <i>Mycobacterium leprae</i> in slit skin smear (SSS) is a gold standard technique for the leprosy diagnosis. Over recent years, molecular diagnosis by using PCR has been increasingly used as an alternative for its diagnosis due to its higher sensitivity. This study was carried out for comparative evaluation of PCR and SSS microscopy in a cohort of new leprosy cases diagnosed in B. P. Koirala Institute of health Sciences, Dharan, Nepal.</p><p>Methodology/Principal Findings</p><p>In this prospective crossectional study, 50 new clinically diagnosed cases of leprosy were included. DNA was extracted from SSS and PCR was carried out to amplify 129 bp sequence of <i>M</i>. <i>leprae</i> repetitive element. Sensitivity of SSS and PCR was 18% and 72% respectively. Improvement of 54% case detection by PCR clearly showed its advantage over SSS. Furthermore, PCR could confirm the leprosy diagnosis in 66% of AFB negative cases indicating its superiority over SSS. In the paucibacillary (PB) patients, whose BI was zero; sensitivity of PCR was 44%, whereas it was 78% in the multibacillary patients.</p><p>Conclusions/Significance</p><p>Our study showed PCR to be more sensitive than SSS microscopy in diagnosing leprosy. Moreover, it explored the characteristic feature of PCR which detected higher level of early stage(PB) cases tested negative by SSS. Being an expensive technique, PCR may not be feasible in all the cases, however, it would be useful in diagnosis of early cases of leprosy as opposed to SSS.</p></div

Agarose gel image of the <i>M</i>. <i>leprae</i> PCR products.

<p>M; Molecular markers. Lanes 1–5; Patient samples. L6; Negative control. L7; Positive control.</p

Sensitivity of Microscopy and PCR with Confidence interval (CI).

<p>Sensitivity of Microscopy and PCR with Confidence interval (CI).</p

Flow chart of overview of methods.

<p>Flow chart of overview of methods.</p

ROC curve of PCR diagnosis with RJ classification.

<p>ROC curve of PCR diagnosis with RJ classification.</p

Correlation of WHO clinical types, results of skin smears for AFB and PCR.

<p>Correlation of WHO clinical types, results of skin smears for AFB and PCR.</p

Flow diagram of participants.

<p>Flow diagram of participants.</p

Molecular targets of ZAS3 in RANK signaling.

<p>Shown is a schematic diagram depicting the multiple targets of ZAS3 involved in the regulation of RANK signaling, important for osteoclastogenic differentiation and function. Binding of RANKL to RANK activates cytoplasmic ZAS3 that (i) induces expression of TRAF6 and association of ZAS3 and TRAF6 leading to the recruitment of TGF-β-activated kinase 1 (TAK1) binding protein 2/3 (TAB2/3) to polyubiquitinated TRAF6, which in turn activates TAB1/TAK1, inhibitor of NF-kB alpha (IkBα) kinase (IKK) complexes, and nuclear translocation of NF-kB p50/p65; (ii) Simultaneously, ZAS3 activates mitogen-activated protein kinases to activate the transcription factor AP-1 via phosphorylation of p38 and assembly of c-Jun/Fos; (iii) ZAS3 associates with c-Jun to activate AP1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017161#pone.0017161-Oukka2" target="_blank">[13]</a>; (iv) ZAS3 itself can act as a transcription factor, i.e., it translocates into the nucleus and binds to gene regulatory elements to activate transcription of osteoclast-associated genes, such as TRAP and CTSK, and probably NFATc1; and (iv) through the activation of NF-kB, ZAS3 also activates NFATc1 that is shown to integrate sRANKL signaling in the terminal differentiation of osteoclasts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017161#pone.0017161-Asagiri1" target="_blank">[30]</a>. Additionally, ZAS3 mobilizes intracellular calcium, probably through one of its target genes, the calcium binding protein <i>S100A4/mts1 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017161#pone.0017161-Hjelmsoe1" target="_blank">[4]</a>, to activate calcineurin causing the dephosphorylation and nuclear translocation of NFATc1. ZAS3 individually, or in a transcriptional complex in conjunction with NTAFc1 and NF-kB through association with AP-1 drives the transcription program for osteoclast differentiation. Important signaling molecules, transcription factors, or enzymes involved in RANK signaling whose expression levels or protein-protein interactions shown here to be regulated by ZAS3 are indicated with asterisks.</p

The bones of adult <i>ZAS3</i> knockout mice have increased bone strength, thickness, and mineralization.

<p>(A) Biomechanical properties of femurs were evaluated by three-point bending test. Load to fracture (Fx) was significantly increased in both male and female <i>ZAS3−/−</i> mice compared to +/+ and +/− control mice (*, <i>p</i><0.05). (B) & (C) Micro-CT reconstruction images of femora of 4-month-old mice. Bar represents 1 mm. (D) Sagittal section of distal femora from 5-month-old mice stained with von Kossa's method plus MacNeal tetrachrome counterstain. Mineralized bone was stained black and collagen type 1 was stained light blue. Bar represents 1 mm. +/+ <i>ZAS3</i> WT mice, −/− <i>ZAS3</i> KO mice, and +/− heterozygous ZAS3 mice.</p

ZAS3 is essential for RANKL-mediated osteoclastogenesis of bone marrow precursors.

<p>(A) Western blot analysis of total protein lysates prepared from bone marrow macrophages (BMM) cultured without (−) or with (+) sRANKL (50 ng/ml) for 3 days. Bone marrow aspirates isolated from WT mice were cultured in complete medium with M-CSF (50 ng/ml) for 3 days, and non-adherent cells were harvested and further cultured with M-CSF (50 ng/ml) and sRANKL (50 ng/ml) for 3 days. (B) Immunohistochemical analysis of ZAS3 (red) in bone marrow macrophages (BMM) cultured with M-CSF and sRANKL (50 ng/ml each) at an early phase of osteoclastogenesis. White arrow indicates a mononucleated cell in which ZAS3 shows prominently nuclear localization. Yellow arrow highlights the membrane proximity of ZAS3 in a cell that contained two nuclei and therefore, had undergone the first cell fusion. Nuclei were stained with DAPI (blue). (C) TRAP staining of BMM cultured in the presence of M-CSF and sRANKL (50 ng/ml each). More TRAP+ (cells stained red) and OLC were observed from <i>ZAS3+/+</i> than <i>ZAS3−/−</i> BMM at day 4 (upper panels) and day 6 (lower panel). At day 6, most <i>ZAS3+/+</i> BMM had differentiated into large multinucleated TRAP positive cells while <i>ZAS3−/−</i> BMM was inefficient to do so and failed to form large spreading TRAP+ OLC. Scale bar, 100 µm. Data were representatives of 1 out of 3 independent experiments. (D) Bar charts showing the number of TRAP+ multinucleated OLC (more than 3 nuclei) formed in response to sRANKL and M-CSF stimulation (initially plated 5000 cells per well of an 8 well slide). Data were expressed as the mean ± SD from 3 independent experiments. (E) Western blot analysis of protein lysates of BMM cultured with M-CSF and sRANKL for 6 days with indicated antibodies. +/+ <i>ZAS3</i> WT mice, −/− <i>ZAS3</i> KO mice, and +/− heterozygous ZAS3 mice.</p