Clinical Diagnosis and Laboratory Testing of Abnormal Appearing Toenails: A Retrospective Assessment of Confirmatory Testing for Onychomycosis in the United States, 2022-2023.

Onychomycosis is an under-recognized healthcare burden. Despite the risk of misdiagnosis, confirmatory laboratory testing is under-utilized. Histopathologic examination with polymerase chain reaction (PCR) is currently the most effective diagnostic method; it offers direct detection and identification of a fungal invasion. In this retrospective cohort study, we assessed confirmatory testing results, with matching clinical diagnoses, in 96,293 nail specimens submitted during a 9-month period from 2022 to 2023. Toenail specimens were examined using fungal culture, histopathology and/or PCR. Clinical diagnoses were identified using the International Classification of Diseases 10th Revision codes. For clinically diagnosed onychomycosis patients, the overall positivity rate was 59.4%; a similar positivity rate (59.5%) was found in patients with clinically diagnosed non-fungal nail dystrophy. Performing a histopathologic examination with PCR was more likely to provide pathogen identification results than using fungal culture. Male patients had a higher rate of onychomycosis overall; however, female patients had more non-dermatophyte mold onychomycosis caused by Aspergillus. Clinically diagnosed onychomycosis patients with a co-diagnosis of tinea pedis were more likely to test positive for onychomycosis by PCR (odds ratio [OR]: 4.2; 95% confidence interval [CI]: 2.7-6.4), histopathology (OR: 2.5; 95% CI: 2.0-3.1) and fungal culture (OR: 3.2; 95% CI: 1.5-6.6). Our results support the use of confirmatory laboratory testing when there is a clinical diagnosis of onychomycosis.

Antifungal Resistance, Susceptibility Testing and Treatment of Recalcitrant Dermatophytosis Caused by Trichophyton indotineae: A North American Perspective on Management.

There is an ongoing epidemic of chronic, relapsing dermatophytoses caused by Trichophyton indotineae that are unresponsive to one or multiple antifungal agents. Although this new species may have originated from the Indian subcontinent, there has been a notable increase of its reporting in other countries. Based on current literature, antifungal susceptibility testing (AFST) showed a large variation of terbinafine minimum inhibitory concentrations (MICs) (0.04 to ≥ 32 µg/ml). Elevated terbinafine MICs can be attributed to mutations in the squalene epoxidase gene (single mutations: Leu393Phe, Leu393Ser, Phe397Leu, and double mutations: Leu393Phe/Ala448Thr, Phe397Leu/Ala448Thr). Itraconazole MICs had a lower range when compared with that of terbinafine (0.008-16 µg/ml, with most MICs falling between 0.008 µg/ml and < 1 µg/ml). The interpretation of AFST results remains challenging due to protocol variations and a lack of established breakpoints. Adoption of molecular methods for resistance detection, coupled with AFST, may provide a better evaluation of the in vitro resistance status of T. indotineae. There is limited information on treatment options for patients with confirmed T. indotineae infections by molecular diagnosis; preliminary evidence generated from case reports and case series points to itraconazole as an effective treatment modality, while terbinafine and griseofulvin are generally not effective. For physicians working outside of endemic regions, there is currently an unmet need for standardized clinical trials to establish treatment guidelines; in particular, combination therapy of oral and topical agents (e.g., itraconazole and ciclopirox), as well as with other azoles (i.e., fluconazole, voriconazole, ketoconazole), warrants further investigation as multidrug resistance is a possibility for T. indotineae.

Single-Point Nail Sampling to Diagnose Onychomycosis Caused by Non-Dermatophyte Molds: Utility of Polymerase Chain Reaction (PCR) and Histopathology.

The three most commonly used methods for diagnosing non-dermatophyte mold (NDM) onychomycosis are culture, polymerase chain reaction (PCR), and histopathology. Toenail samples from 512 patients (1 sample/patient) with suspected onychomycosis were examined using all three diagnostic tests. A statistically significant association was found between PCR and histopathology results, as well as between fungal culture and histopathology results. All PCR-positive and culture-positive dermatophyte samples were confirmed by histopathology. However, 15/116 (12.9%) of culture-positive NDM samples had negative histopathology results, while all PCR-positive NDM samples were confirmed by histopathology. The overall rate of dermatophyte detection was higher using PCR compared to culture (38.9% vs. 11.7%); the lower rate of NDM detection by PCR (11.7% vs. 38.9%) could be attributed to the restriction of the assay design to seven pre-selected targets. When repeat sampling in the clinic is not possible, a combination of NDM detection by PCR and positive histopathology of hyphae may be a proxy for NDM infection, particularly where the NDM occurs without a concomitant dermatophyte. There was a high degree of correlation between negative PCR and negative histopathology. A negative PCR result with negative histopathology findings may be a reliable proxy for the diagnosis of non-fungal dystrophy.

Detection of Squalene Epoxidase Mutations in United States Patients with Onychomycosis: Implications for Management.

Resistance to oral terbinafine, the most commonly used antifungal to treat dermatophytosis and onychomycosis worldwide, is being increasingly reported. In this study, we aimed to investigate the species distribution and prevalence of squalene epoxidase mutations among toenail dermatophyte isolates. Samples from 15,683 patients suspected of onychomycosis visiting the offices of dermatologists and podiatrists in the United States were analyzed. Clinical information was reviewed, and dermatophyte species with or without squalene epoxidase mutations were detected using multiplex real-time PCRs. The frequency of dermatophytes was 37.6%; of isolates belonging to the Trichophyton genus, 88.3% were the T. rubrum complex, and 11.2% were the T. mentagrophytes complex. Individuals aged >70 years exhibited higher infection rates for the T. mentagrophytes complex. The overall mutation rate among Trichophyton spp. was 3.7%, with a higher mutation rate detected in the T. mentagrophytes complex (4.3 vs. 3.6%). Commonly detected mutations were T1189C/Phe397Leu (34.5%), T1306C/Phe415Ser (16.0%), and C1191A/Phe397Leu (11.0%). Squalene epoxidase gene mutations associated with decreased terbinafine susceptibility have been identified in United States patients with toenail onychomycosis. Physicians should be aware of the risk factors for resistance development and engage in antifungal stewardship practices such as directed diagnosis and treatment of dermatophytosis and onychomycosis.

Preferential Retention and Transport of Perfluorooctanesulfonic Acid in a Dolomite Aquifer.

Per- and polyfluoroalkyl substances (PFAS) can represent a significant human health risk if present in aquifers used as a drinking water source. Accurate assessment of PFAS exposure risks requires an improved understanding of field-scale PFAS transport in groundwater. Activities at a former firefighter training site in University Park, Pennsylvania introduced perfluorooctanesulfonic acid (PFOS) to the underlying dolomite aquifer. Groundwater sampling from 2015 to 2018 delineated a PFOS plume with two concentration maxima located approximately 20 and approximately 220 m downgradient of the training site, separated by a zone of lower concentrations. We use a combination of analytical and numerical models, informed by independent measurements of aquifer porosity, hydraulic conductivity, and organic carbon content, to interpret the field observations. Our analysis demonstrates that preferential retention and transport resulting from simple heterogeneity in bedrock sorption, as caused by organic carbon (OC) content variability, provides a plausible explanation for plume separation. Dissolved PFOS partitions strongly to organic solids (high Koc ), so even a small OC (<1 wt%) significantly retards PFOS transport, whereas zones with little to no OC allow for transport rates that approximate those of a conservative solute. Our work highlights an important consideration for modeling the groundwater transport of PFOS, and other compounds with high Koc . In aquifers with discrete layers of varying OC, models using a uniform site-average OC will underestimate transport distances, thereby misrepresenting exposure risks for downgradient communities.

Lipid Biomarker Record of the Serpentinite-Hosted Ecosystem of the Samail Ophiolite, Oman and Implications for the Search for Biosignatures on Mars.

Serpentinization is a weathering process in which ultramafic rocks react with water, generating a range of products, including serpentine and other minerals, in addition to H2 and low-molecular-weight hydrocarbons that are capable of sustaining microbial life. Lipid biomarker analyses of serpentinite-hosted ecosystems hold promise as tools for investigating microbial activity in ancient Earth environments and other terrestrial planets such as Mars because lipids have the potential for longer term preservation relative to DNA, proteins, and other more labile organic molecules. Here, we report the first lipid biomarker record of microbial activity in the mantle section of the Samail Ophiolite, in the Sultanate of Oman, a site undergoing active serpentinization. We detected isoprenoidal (archaeal) and branched (bacterial) glycerol dialkyl glycerol tetraether (GDGT) lipids, including those with 0-3 cyclopentane moieties, and crenarchaeol, an isoprenoidal GDGT containing four cyclopentane and one cyclohexane moieties, as well as monoether lipids and fatty acids indicative of sulfate-reducing bacteria. Comparison of our geochemical data and 16S rRNA data from the Samail Ophiolite with those from other serpentinite-hosted sites identifies the existence of a common core serpentinization microbiome. In light of these findings, we also discuss the preservation potential of serpentinite lipid biomarker assemblages on Earth and Mars. Continuing investigations of the Samail Ophiolite and other terrestrial analogues will enhance our understanding of microbial habitability and diversity in serpentinite-hosted environments on Earth and elsewhere in the Solar System.

Viable cyanobacteria in the deep continental subsurface.

Cyanobacteria are ecologically versatile microorganisms inhabiting most environments, ranging from marine systems to arid deserts. Although they possess several pathways for light-independent energy generation, until now their ecological range appeared to be restricted to environments with at least occasional exposure to sunlight. Here we present molecular, microscopic, and metagenomic evidence that cyanobacteria predominate in deep subsurface rock samples from the Iberian Pyrite Belt Mars analog (southwestern Spain). Metagenomics showed the potential for a hydrogen-based lithoautotrophic cyanobacterial metabolism. Collectively, our results suggest that they may play an important role as primary producers within the deep-Earth biosphere. Our description of this previously unknown ecological niche for cyanobacteria paves the way for models on their origin and evolution, as well as on their potential presence in current or primitive biospheres in other planetary bodies, and on the extant, primitive, and putative extraterrestrial biospheres.

The influence of pressure on crude oil biodegradation in shallow and deep Gulf of Mexico sediments.

A significant portion of oil released during the Deepwater Horizon disaster reached the Gulf of Mexico (GOM) seafloor. Predicting the long-term fate of this oil is hindered by a lack of data about the combined influences of pressure, temperature, and sediment composition on microbial hydrocarbon remineralization in deep-sea sediments. To investigate crude oil biodegradation by native GOM microbial communities, we incubated core-top sediments from 13 GOM sites at water depths from 60-1500 m with crude oil under simulated aerobic seafloor conditions. Biodegradation occurred in all samples and followed a predictable compound class sequence dictated by molecular weight and structure. 45 to ~100% of total n-alkane and 3 to 60% of total polycyclic aromatic hydrocarbons (PAH) were depleted. In reactors incubated at 4°C and at pressures of 6-15 MPa, the depletion in total n-alkane was inversely correlated to pressure (R2 ~ 0.85), equivalent to a 4% decrease in total n-alkane depletion for every 1 MPa increase. Our results indicated a modest inhibitory effect of pressure on biodegradation over our experimental range. However, the expansion of oil exploration to deeper waters (e.g., 5000 m) opens the risk of spills at conditions at which pressure might have a more pronounced effect.

Draft Genome Sequence of the Piezotolerant and Crude Oil-Degrading Bacterium Rhodococcus qingshengii Strain TUHH-12.

We report here the draft genome sequence of Rhodococcus qingshengii strain TUHH-12. The ability of this piezotolerant bacterium to grow on crude oil and tetracosane as sole carbon sources at 150 × 10(5) Pa makes it useful in studies of hydrocarbon degradation under simulated deep-sea conditions.

Planktonic Euryarchaeota are a significant source of archaeal tetraether lipids in the ocean.

Archaea are ubiquitous in marine plankton, and fossil forms of archaeal tetraether membrane lipids in sedimentary rocks document their participation in marine biogeochemical cycles for >100 million years. Ribosomal RNA surveys have identified four major clades of planktonic archaea but, to date, tetraether lipids have been characterized in only one, the Marine Group I Thaumarchaeota. The membrane lipid composition of the other planktonic archaeal groups--all uncultured Euryarchaeota--is currently unknown. Using integrated nucleic acid and lipid analyses, we found that Marine Group II Euryarchaeota (MG-II) contributed significantly to the tetraether lipid pool in the North Pacific Subtropical Gyre at shallow to intermediate depths. Our data strongly suggested that MG-II also synthesize crenarchaeol, a tetraether lipid previously considered to be a unique biomarker for Thaumarchaeota. Metagenomic datasets spanning 5 y indicated that depth stratification of planktonic archaeal groups was a stable feature in the North Pacific Subtropical Gyre. The consistent prevalence of MG-II at depths where the bulk of exported organic matter originates, together with their ubiquitous distribution over diverse oceanic provinces, suggests that this clade is a significant source of tetraether lipids to marine sediments. Our results are relevant to archaeal lipid biomarker applications in the modern oceans and the interpretation of these compounds in the geologic record.