Evaluation of qPCR-Based Assays for Leprosy Diagnosis Directly in Clinical Specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations

Effect of acute hypoxia on respiratory muscle fatigue in healthy humans

<p>Abstract</p> <p>Background</p> <p>Greater diaphragm fatigue has been reported after hypoxic versus normoxic exercise, but whether this is due to increased ventilation and therefore work of breathing or reduced blood oxygenation per se remains unclear. Hence, we assessed the effect of different blood oxygenation level on isolated hyperpnoea-induced inspiratory and expiratory muscle fatigue.</p> <p>Methods</p> <p>Twelve healthy males performed three 15-min isocapnic hyperpnoea tests (85% of maximum voluntary ventilation with controlled breathing pattern) in normoxic, hypoxic (SpO₂= 80%) and hyperoxic (FiO₂= 0.60) conditions, in a random order. Before, immediately after and 30 min after hyperpnoea, transdiaphragmatic pressure (P_di,tw) was measured during cervical magnetic stimulation to assess diaphragm contractility, and gastric pressure (P_ga,tw) was measured during thoracic magnetic stimulation to assess abdominal muscle contractility. Two-way analysis of variance (time x condition) was used to compare hyperpnoea-induced respiratory muscle fatigue between conditions.</p> <p>Results</p> <p>Hypoxia enhanced hyperpnoea-induced P_di,twand P_ga,twreductions both immediately after hyperpnoea (P_di,tw: normoxia -22 ± 7% vs hypoxia -34 ± 8% vs hyperoxia -21 ± 8%; P_ga,tw: normoxia -17 ± 7% vs hypoxia -26 ± 10% vs hyperoxia -16 ± 11%; all <it>P </it>< 0.05) and after 30 min of recovery (P_di,tw: normoxia -10 ± 7% vs hypoxia -16 ± 8% vs hyperoxia -8 ± 7%; P_ga,tw: normoxia -13 ± 6% vs hypoxia -21 ± 9% vs hyperoxia -12 ± 12%; all <it>P </it>< 0.05). No significant difference in P_di,twor P_ga,twreductions was observed between normoxic and hyperoxic conditions. Also, heart rate and blood lactate concentration during hyperpnoea were higher in hypoxia compared to normoxia and hyperoxia.</p> <p>Conclusions</p> <p>These results demonstrate that hypoxia exacerbates both diaphragm and abdominal muscle fatigability. These results emphasize the potential role of respiratory muscle fatigue in exercise performance limitation under conditions coupling increased work of breathing and reduced O₂transport as during exercise in altitude or in hypoxemic patients.</p

A diarylamine derived from anthranilic acid inhibits ZIKV replication

Zika virus (ZIKV) is a mosquito-transmitted Flavivirus, originally identified in Uganda in 1947 and recently associated with a large outbreak in South America. Despite extensive efforts there are currently no approved antiviral compounds for treatment of ZIKV infection. Here we describe the antiviral activity of diarylamines derived from anthranilic acid (FAMs) against ZIKV. A synthetic FAM (E3) demonstrated anti-ZIKV potential by reducing viral replication up to 86%. We analyzed the possible mechanisms of action of FAM E3 by evaluating the intercalation of this compound into the viral dsRNA and its interaction with the RNA polymerase of bacteriophage SP6. However, FAM E3 did not act by these mechanisms. In silico results predicted that FAM E3 might bind to the ZIKV NS3 helicase suggesting that this protein could be one possible target of this compound. To test this, the thermal stability and the ATPase activity of the ZIKV NS3 helicase domain (NS3Hel) were investigated in vitro and we demonstrated that FAM E3 could indeed bind to and stabilize NS3Hel