Pathology and Monkeypox virus Localization in Tissues From Immunocompromised Patients With Severe or Fatal Mpox.

BACKGROUND: Pathology and Monkeypox virus (MPXV) tissue tropism in severe and fatal human mpox is not thoroughly described but can help elucidate the disease pathogenesis and the role of coinfections in immunocompromised patients. METHODS: We analyzed biopsy and autopsy tissues from 22 patients with severe or fatal outcomes to characterize pathology and viral antigen and DNA distribution in tissues by immunohistochemistry and in situ hybridization. Tissue-based testing for coinfections was also performed. RESULTS: Mucocutaneous lesions showed necrotizing and proliferative epithelial changes. Deceased patients with autopsy tissues evaluated had digestive tract lesions, and half had systemic tissue necrosis with thrombotic vasculopathy in lymphoid tissues, lung, or other solid organs. Half also had bronchopneumonia, and one-third had acute lung injury. All cases had MPXV antigen and DNA detected in tissues. Coinfections were identified in 5 of 16 (31%) biopsy and 4 of 6 (67%) autopsy cases. CONCLUSIONS: Severe mpox in immunocompromised patients is characterized by extensive viral infection of tissues and viremic dissemination that can progress despite available therapeutics. Digestive tract and lung involvement are common and associated with prominent histopathological and clinical manifestations. Coinfections may complicate mpox diagnosis and treatment. Significant viral DNA (likely correlating to infectious virus) in tissues necessitates enhanced biosafety measures in healthcare and autopsy settings.

Antagonism between substitutions in ß-lactamase explains a path not taken in the evolution of bacterial drug resistance.

CTX-M ß-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-µs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

Comparison of Upper Respiratory Viral Load Distributions in Asymptomatic and Symptomatic Children Diagnosed with SARS-CoV-2 Infection in Pediatric Hospital Testing Programs.

The distribution of upper respiratory viral loads (VL) in asymptomatic children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. We assessed PCR cycle threshold (Ct) values and estimated VL in infected asymptomatic children diagnosed in nine pediatric hospital testing programs. Records for asymptomatic and symptomatic patients with positive clinical SARS-CoV-2 tests were reviewed. Ct values were (i) adjusted by centering each value around the institutional median Ct value from symptomatic children tested with that assay and (ii) converted to estimated VL (numbers of copies per milliliter) using internal or manufacturer data. Adjusted Ct values and estimated VL for asymptomatic versus symptomatic children (118 asymptomatic versus 197 symptomatic children aged 0 to 4 years, 79 asymptomatic versus 97 symptomatic children aged 5 to 9 years, 69 asymptomatic versus 75 symptomatic children aged 10 to 13 years, 73 asymptomatic versus 109 symptomatic children aged 14 to 17 years) were compared. The median adjusted Ct value for asymptomatic children was 10.3 cycles higher than for symptomatic children (P < 0.0001), and VL were 3 to 4 logs lower than for symptomatic children (P < 0.0001); differences were consistent (P < 0.0001) across all four age brackets. These differences were consistent across all institutions and by sex, ethnicity, and race. Asymptomatic children with diabetes (odds ratio [OR], 6.5; P = 0.01), a recent contact (OR, 2.3; P = 0.02), and testing for surveillance (OR, 2.7; P = 0.005) had higher estimated risks of having a Ct value in the lowest quartile than children without, while an immunocompromised status had no effect. Children with asymptomatic SARS-CoV-2 infection had lower levels of virus in their nasopharynx/oropharynx than symptomatic children, but the timing of infection relative to diagnosis likely impacted levels in asymptomatic children. Caution is recommended when choosing diagnostic tests for screening of asymptomatic children.

Synergistic effects of functionally distinct substitutions in ß-lactamase variants shed light on the evolution of bacterial drug resistance.

The CTX-M ß-lactamases have emerged as the most widespread extended-spectrum ß-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103-106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103-106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 ß-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a "switch" that toggles the conformations of the 103-106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M-like, cefotaximase-favorable conformation.

Comparison of Bruker Biotyper® and Vitek® MS matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry platforms for the identification of filamentous fungi.

Aims: To evaluate the performance of two matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry platforms to identify molds isolated from clinical specimens. Methods: Fifty mold isolates were analyzed on Bruker Biotyper® and Vitek® MS platforms. Two Bruker Biotyper extraction protocols were assessed alongside the US FDA-approved extraction protocol for Vitek MS. Results: The Bruker Biotyper modified NIH-developed extraction protocol correctly identified more isolates than Bruker's protocol (56 vs 33%). For species in the manufacturers' databases, Vitek MS correctly identified 85% of isolates, with 8% misidentifications. The Bruker Biotyper identified 64%, with no misidentifications. For isolates not in the databases, the Bruker Biotyper did not misidentify any, and Vitek MS misidentified 36%. Conclusion: Both the Vitek MS and Bruker Biotyper accurately identified the fungal isolates, however Vitek MS was more likely to misidentify isolates than the Bruker Biotyper.

There are two different mass spectrometry systems that can be used in the hospital laboratory to find out what kind of mold is growing from a patient sample: the Vitek^® MS and Bruker Biotyper^® systems. This study compared how well they work for mold identification and also looked at two different ways to prepare the mold for testing. The Vitek MS system identified more molds, but also made more mistakes when identifying them. The Bruker Biotyper identified fewer molds but did not make any mistakes on the identification. The Vitek MS system sometimes gets the type of mold wrong, so more tests may be needed to be sure of the result. The Bruker Biotyper is more accurate because it got all of the molds correct, but it could not identify as many.

Novel use of FDA-approved drugs identified by cluster analysis of behavioral profiles.

Repurposing FDA-approved drugs is an efficient and cost-effective approach in the development of therapeutics for a broad range of diseases. However, prediction of function can be challenging, especially in the brain. We screened a small-molecule library with FDA-approved drugs for effects on behavior. The studies were carried out using zebrafish larvae, imaged in a 384-well format. We found that various drugs affect activity, habituation, startle responses, excitability, and optomotor responses. The changes in behavior were organized in behavioral profiles, which were examined by hierarchical cluster analysis. One of the identified clusters includes the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506), which are immunosuppressants and potential therapeutics in the prevention of Alzheimer's disease. The calcineurin inhibitors form a functional cluster with seemingly unrelated drugs, including bromocriptine, tetrabenazine, rosiglitazone, nebivolol, sorafenib, cabozantinib, tamoxifen, meclizine, and salmeterol. We propose that drugs with 'CsA-type' behavioral profiles are promising candidates for the prevention and treatment of Alzheimer's disease.

Performance of six SARS-CoV-2 RNA detection systems in symptomatic and asymptomatic pediatric and maternal patients.

Aim: This study evaluated the real-world performance of six test systems for detection of SARS-CoV-2 in 138 pediatric and 110 adult maternal patients. Materials & methods: Nasopharyngeal swabs were tested directly using the Aptima™ SARS-CoV-2 (Aptima) and Simplexa™ COVID-19 Direct (Simplexa), and with Altona RealStar® RT-PCR and CDC RT-PCR with nucleic acid extracted on the Roche® MagNA Pure 96 (Altona-MP96) or bioMérieux EMAG® (Altona-EMAG). Results/Conclusion: Overall percent-positive and percent-negative agreements among the six test systems were, respectively: Aptima: 94.8 and 100%; Altona-MP96: 96.5 and 99.3%; CDC-MP96: 100 and 99.3%; Altona-EMAG: 86.1 and 100%; CDC-EMAG: 98.2 and 100%; Simplexa: 87 and 99.2%. The six test systems showed agreement ranging from 92.7 (κ = 0.85) to 98.8% (κ = 0.98).

Comparison of Mid-Turbinate and Nasopharyngeal Specimens for Molecular Detection of SARS-CoV-2 Among Symptomatic Outpatients at a Pediatric Drive-Through Testing Site.

BACKGROUND: Nasopharyngeal (NP) specimen testing by reverse transcriptase polymerase chain reaction (RT-PCR) is the standard of care for detecting SARS-CoV-2. Data comparing the sensitivity and specificity of the NP specimen to the less invasive, mid-turbinate (MT) nasal specimen in children are limited. METHODS: Paired clinical NP and research MT specimens were collected from children <18 years with respiratory symptoms and tested by molecular assays to detect SARS-CoV-2 RNA. Sensitivity, specificity, and agreement (Cohen's kappa [κ]) were calculated for research MT specimens compared to the clinical NP specimens. RESULTS: Out of 907 children, 569 (62.7%) had parental consent and child assent when appropriate to participate and provided paired MT and NP specimens a median of 4 days after symptom onset (range 1-14 days). 16.5% (n = 94) of MT specimens were positive for SARS-CoV-2 compared with 20.0% (n = 114) of NP specimens. The sensitivity of research MT compared to clinical NP specimens was 82.5% (95% CI: 74.2%, 88.9%), specificity was 100.0% (95% CI: 99.2%, 100.0%), and overall agreement was 96.1% (κ = 0.87). The sensitivity of MT specimens decreased with time from 100% (95% CI: 59.0%, 100.0%) on day 1 of illness to 82.1% (95% CI: 73.8%, 88.7%) within 14 days of illness onset; sensitivity was generally >90% when specimens were collected within the first week of illness. CONCLUSION: MT specimens, particularly those collected within the first week of illness, have moderately reduced sensitivity and equivalent specificity to less-tolerated NP specimens in pediatric outpatients. MT specimen use in children may represent a viable alternative to NP specimen collection.