A competitive activity-based protein profiling platform yields cell wall synthesis inhibitors active against replicating and non-replicating Mycobacterium tuberculosis

ABSTRACTThe identification and validation of a small molecule’s targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule’s targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here we studied the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening ~200 compounds, we focused on two inhibitors active against both exponentially replicating and hypoxia-induced drug-tolerant Mtb that form part of a four-compound structure-activity series. The compound with negligible activity revealed potential false positive targets not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirmed that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings showed that ABPP identifies the targets most likely relevant to a compound’s antibacterial activity.

Performance of Xpert MTB/RIF Ultra for diagnosis of pulmonary and extra-pulmonary tuberculosis, one year of use in a multi-centric hospital laboratory in Brussels, Belgium

Among the challenges in controlling tuberculosis, a rapid and accurate diagnostic test for the detection of Mycobacterium tuberculosis complex (MTBc) and its resistance to first line therapies is crucial. We evaluated the performance of the Xpert MTB/RIF Ultra assay (Xpert Ultra) for the rapid detection of MTBc and rifampicin resistance (RR) in 1120 pulmonary and 461 extra-pulmonary clinical specimens and compared it with conventional phenotypic techniques. The Xpert Ultra assay detected MTBc in 223 (14.1%) samples with an overall sensitivity and specificity, using culture as the “gold standard”, of 91.1% (95% CI, 85.6–95.1) and 94.5% (95% CI, 93.1–95.6), respectively. The sensitivity of the Xpert Ultra test for smear-negative extra-pulmonary specimens was high (87.1%), even higher than with smear-negative pulmonary specimens (81.8%). But this enhanced sensitivity came with a low overall specificity of smear-negative extra-pulmonary specimens (66.7%). For 73 patients, 79/1423 (3.4%) negative mycobacterial culture samples were found to be positive with Xpert Ultra. Clinical data was necessary to correctly interpret potential false-positive results, especially trace-positive results. Sensitivity of the Xpert Ultra to detect RR compared to drug susceptibility testing was 100% (95% CI, 29.2–100) and specificity was 99.2% (95% CI, 95.8–100). We concluded that the Xpert Ultra test is able to provide a reliable TB diagnosis within a significantly shorter turnaround time than culture. This is especially true for paucibacillary samples such as smear-negative pulmonary specimens and extra-pulmonary specimens.

Potential False-Positive and False-Negative Results for COVID-19 IgG/IgM Antibody Testing After Heat-Inactivation

Objectives: With the worldwide spread of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), various antibody detection kits have been developed to test for SARS-CoV-2– specific IgG, IgM, and total antibody. However, the use of different testing methods under various heat-inactivation conditions might affect the COVID-19 detection results.Methods: Seven different antibody detection kits produced by four manufacturers for detection of SARS-CoV-2 IgG, IgM, and total antibody were tested at Wuhan Huoshenshan Hospital, China. Most of the kits used the indirect immunity, capture, and double-antigen sandwich methods. The effects of various heat-inactivation conditions on SARS-CoV-2-specific IgG, IgM, and total antibody detection were analyzed for the different test methods.Results: Using the indirect immunity method, values for SARS-CoV-2 IgG antibody significantly increased and those for IgM antibody decreased with increasing temperature of heat-inactivation using indirect immunity method. However, values for SARS-CoV-2 IgM and total antibody showed no change when the capture and double-antigen sandwich methods were used. The changes in IgG and IgM antibody values with the indirect immunity method indicated that heat-inactivation could affect COVID-19 detection results obtained using this method. In particular, 18 (22.2%) SARS-CoV-2 IgM positive samples were detected as negative with heat-inactivation at 65°C for 30 min, and one (25%) IgG negative sample was detected as positive after heat-inactivation at 56°C for 60 min and 60°C for 30 min.Conclusions: Heat-inactivation could increase SARS-CoV-2 IgG antibody values, and decrease IgM antibody values, causing potential false-positive or false-negative results for COVID-19 antibody detection using the indirect immunity method. Thus, before conducting antibody testing, the testing platforms should be evaluated in accordance with the relevant requirements to ensure accurate COVID-19 detection results.

Seminal Vesicle Adherent to Rectal Wall Following Neoadjuvant Chemoradiotherapy: A Potential False-Positive Diagnostic Pitfall

The standard of care for stage T3 and stage T4 rectal adenocarcinomas involves neoadjuvant chemoradiotherapy followed by either low anterior resection or abdominopelvic resection. The presence of residual adenocarcinoma or positive surgical margins provides useful prognostic information and can influence ongoing adjuvant therapy. Although uncommon, mimics of treated adenocarcinoma may be present in the surgical specimen. A high index of suspicion is critical in avoiding potential false-positive pitfalls, and the exclusion of mimics of treated adenocarcinoma is paramount to accurate diagnosis and treatment. Seminal vesicle epithelium has long been a challenge in differentiating prostatic adenocarcinoma from benign epithelium. However, the role of incidental seminal vesiculectomy in rectal resections due to fibrous adhesion to the rectal wall secondary to chemoradiation has not been studied. As the seminal vesicle epithelium can show markedly atypical nuclei with radiation-type effect at baseline, the potential risk of misinterpretation as residual adenocarcinoma is high. In this article, we present 2 case reports of rectal adenocarcinoma treated with neoadjuvant chemoradiotherapy followed by transabdominal resection (low anterior resection or abdominopelvic resection) with incidental seminal vesiculectomies mimicking either residual adenocarcinoma or residual adenocarcinoma at a margin of resection.

Keratoconus: The Masquerade.

We describe the case of an 11-year-old girl with a background of anisometropia and corneal scarring who had a misleading appearance of keratoconus. The patient initially presented at age 6 with a unilateral reduction of visual acuity (VA) in her left eye (6/15). There was also evidence of left corneal scarring which appeared chronic; possibly secondary to previous herpes simplex keratitis (HSK). She was treated with glasses and though her vision initially stabilized (VA 6/6 in both eyes), she was lost to follow-up due to multiple missed appointments. Her vision remained stable for 5 years before she presented with another reduction of VA in her left eye (6/48). The patient was investigated using Pentacam which showed ectatic changes suggestive of keratoconus. There was also progression of the corneal scar in her left eye. With an impression of active stromal herpetic keratitis, the patient was commenced on 200 mg Acyclovir TDS and Dexamethasone minims TDS. 2 months later, her VA improved to 6/7.5 in the left eye. Pentacam was then repeated and the astigmatism, as well as anterior steepening values had reduced. Considering this, clearly HSK had led to a misleading appearance of keratoconus upon initial imaging. This case highlights the potential false positive diagnosis of keratoconus that can occur in younger patients with HSK. Although pentacam results showed advanced ectasia, one should remain mindful of differential diagnoses; particularly seeking out any signs of herpetic eye disease and keeping a low threshold for appropriate treatment.

Characterization of an Amphetamine Interference from Gabapentin in an LC–HRMS Method

An amphetamine interference was observed during the development of an liquid chromatography–high-resolution mass spectrometry (LC–HRMS) multi-class confirmation method for the determination of 47 drugs and metabolites in urine. The interference passed all qualitative criteria for amphetamine leading to potential false-positive results. Upon investigation, it was found that the amphetamine interference was correlated with the presence of high levels of gabapentin. Gabapentin is routinely detected in patient urine specimens at levels in excess of 1 mg/mL as it is widely prescribed at high doses and does not undergo significant metabolism. The source of the interference was identified as a gabapentin in-source fragment isomeric with protonated amphetamine. Here we describe the characterization of this interference and how its effect was mitigated in the LC–HRMS method.