Mechanisms of Staphylococcus aureus survival of trimethoprim-sulfamethoxazole-induced thymineless death.

Trimethoprim-sulfamethoxazole (SXT) is commonly used to treat diverse Staphylococcus aureus infections, including those associated with cystic fibrosis (CF) pulmonary disease. Studies with Escherichia coli found that SXT impairs tetrahydrofolate production, leading to DNA damage, stress response induction, and accumulation of reactive oxygen species (ROS) in a process known as thymineless death (TLD). TLD survival can occur through the uptake of exogenous thymidine, countering the effects of SXT; however, a growing body of research has implicated central metabolism as another potentially important determinant of bacterial survival of SXT and other antibiotics. Here, we conducted studies to better understand the mechanisms of TLD survival in S. aureus. We found that thymidine abundances in CF sputum were insufficient to prevent TLD of S. aureus, highlighting the importance of alternative survival mechanisms in vivo. In S. aureus cultured in vitro with SXT and low thymidine, we frequently identified adaptive mutations in genes encoding carbohydrate, nucleotide, and amino acid metabolism, supporting reduced metabolism as a common survival mechanism. Although intracellular ROS levels rose with SXT treatment in vitro, survival was not improved in the presence of ROS scavengers, unlike in E. coli. SXT challenge induced the SOS response, which was alleviated by added thymidine. Finally, an inactivating mutation in the phosphotransferase gene ptsI conferred both limitation in cellular ATP and improved survival against TLD. Collectively, these results suggest that alterations in core metabolic functions, particularly those that reduce ATP levels, predominantly confer S. aureus survival and persistence during SXT treatment, potentially identifying novel targets for co-treatment.IMPORTANCEStaphylococcus aureus is a ubiquitous organism and one of the leading causes of human infections, many of which are difficult to treat due to persistence, antibiotic resistance, or antibiotic tolerance. As our arsenal of effective antibiotics dwindles, the need for improved treatments becomes increasingly urgent, necessitating a better understanding of the precise mechanisms by which pathogens evade our most critical antimicrobial agents. Here, we report a systematic characterization of the mechanisms of S. aureus survival to treatment with the first-line antistaphylococcal antibiotic trimethoprim-sulfamethoxazole, identifying pathways and candidate targets for enhancing the efficacy of available antimicrobial agents.

Descriptive and molecular epidemiology of leishmaniasis diagnosed from clinical samples in the United States, 2021-2022.

Leishmaniasis is a rare disease in the United States, with an estimated annual incidence of dozens of cases occurring primarily in travelers, migrants, and military personnel. True disease incidence is unknown, since leishmaniasis is not a nationally notifiable condition. Here, we describe the results of molecular leishmaniasis over a 1-year interval (September 2021 to August 2022) when our laboratory served as the primary national reference laboratory for molecular diagnosis of civilian leishmaniasis. We tested 218 specimens submitted from 36 states yielding 94 of the 186 (50.5%) positive cases with species or species complex-level identification and 18 novel mini-exon alleles. Most species belonged to subgenus Viannia (75.6%) and associated with cutaneous or mucocutaneous disease. Cases were associated with recent travel (18.1%), travel timing unspecified (7.4%), migration (7.4%), remote travel (2.1%), military (1.1%), or unknown history (63.8%). These data illustrate the clinical utility of molecular testing for leishmaniasis and provide unique insight into disease epidemiology. IMPORTANCE: Leishmaniasis is a disfiguring, neglected parasitic infection endemic to the Southern United States and the Americas. Despite significant populations at risk-travelers, military and foreign service members, and migrating persons-the epidemiology of the disease in the United States is poorly understood. Moreover, few clinical laboratories in the United States can test for the disease. Here, we present results from 1 year of testing for this disease at a major reference laboratory. These findings are particularly relevant because they coincide with a temporary "pause" on all clinical testing at the CDC. Our findings suggest at least several hundred cases occur each year in the United States. In particular, mucosal leishmaniasis may be more common than previously reported. We also highlight greater genetic diversity in Leishmania species endemic to the Americas than has been previously sampled, with implications for diagnostic specificity.

The Vaginal Microbiome of Transgender and Gender Nonbinary Individuals.

Purpose: The goal of this preliminary study is to describe the vaginal microbiome of transgender and gender nonbinary (TGNB) individuals using nonculture-based techniques. TGNB individuals may undergo gender-affirming surgical procedures, which can include the creation of a neovagina. Little is known about microbial species that comprise this environment in states of health or disease. Methods: In this pilot study, vaginal swabs were self-collected from 15 healthy self-identified TGNB participants (age 26-69 years) and 8 cisgender comparator participants (age 27-50 years) between 2017 and 2018. Next-generation 16S ribosomal RNA sequencing was used to profile individual bacterial communities from all study samples. Results: The TGNB cohort demonstrated significantly higher intraindividual (alpha) diversity than the cisgender group (p=0.0003). Microbial species commensal to the gut and skin were identified only in specimens from TGNB participants. Although Lactobacillus species were dominant in all cisgender comparator samples, they were found at low relative abundance (≤3%) in TGNB samples. Conclusion: In this study, specimens collected from neovaginas showed increased alpha diversity and substantially different composition compared with natal vaginas. In contrast to natal vaginas, neovaginas were not dominated by Lactobacillus, but were hosts to many microbial species. Studies that help to improve our understanding of the neovaginal microbiome may enable clinicians to differentiate between healthy and diseased neovaginal states.

Clinical and in vitro models identify distinct adaptations enhancing Staphylococcus aureus pathogenesis in human macrophages.

Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.

Preventing Surgical Site Infections in the Era of Escalating Antibiotic Resistance and Antibiotic Stewardship.

Importance: According to the Centers for Disease Control and Prevention and governing bodies within the American College of Surgeons, the administration of antibiotics as prophylaxis against infection prior to a planned elective procedure is, with rare exception, routinely recommended. The goal of "getting to zero" infections remains a high priority for policymakers, practitioners, and certainly for patients. Observations: Despite the many advances in surgical technique, skin decontamination, sterile procedure, and enhanced recovery programs, surgical site infections continue to adversely affect procedures as diverse as dental implant surgery, joint arthroplasty, and major abdominal surgery. Although surgical site infection rates are at historically low levels, progress has stalled in recent reporting periods and such infections remain disabling, costly, and occasionally lethal. Stakeholders in the field, including surgeons, infectious diseases specialists, and industry, advocate for strategies emphasizing greater levels of intraoperative sterility or broader-spectrum antibiotic coverage as the most appropriate path forward. Conclusions and Relevance: The current emphasis on ever-increasing levels of intraoperative sterility and extended-spectrum antibiotic use are not sustainable long-term solutions. Continuing to escalate these approaches may contribute to unintended consequences including antimicrobial resistance. Principles of antimicrobial stewardship and microbiome sciences can be applied to inform a more effective and sustainable approach to infection prevention in the field of surgery.

2023 Neer Award for Basic Science: Genetics of Cutibacterium acnes in revision shoulder arthroplasty: a large-scale bacterial whole-genome sequencing study.

BACKGROUND: Cutibacterium acnes is the bacterium most commonly responsible for shoulder periprosthetic joint infection (PJI) and is often cultured from samples obtained at the time of revision for failed shoulder arthroplasty. We sought to determine whether these bacteria originate from the patient or from exogenous sources. We also sought to identify which C. acnes genetic traits were associated with the development of shoulder PJI. METHODS: We performed bacterial whole-genome sequencing of C. acnes from a single-institution repository of cultures obtained before or during primary and revision shoulder arthroplasty and correlated the molecular epidemiology and genetic content of strains with clinical features of infection. RESULTS: A total of 341 isolates collected over a 4-year period from 88 patients were sequenced. C. acnes cultured from surgical specimens demonstrated significant similarity to the strains colonizing the skin of the same patient (P < .001). Infrequently, there was evidence of strains shared across unrelated patients, suggesting that exogenous sources of C. acnes culture-positivity were uncommon. Phylotypes IB and II were modestly associated with clinical features of PJI, but all phylotypes appeared inherently capable of causing disease. Chronic shoulder PJI was associated with the absence of common C. acnes genes involved in bacterial quorum-sensing (luxS, tqsA). CONCLUSION: C. acnes strains cultured from deep intraoperative sources during revision shoulder arthroplasty demonstrate strong genetic similarity to the strains colonizing a patient's skin. Some phylotypes of C. acnes commonly colonizing human skin are modestly more virulent than others, but all phylotypes have a capacity for PJI. C. acnes cultured from cases of PJI commonly demonstrated genetic hallmarks associated with adaptation from acute to chronic phases of infection. This is the strongest evidence to date supporting the role of the patient's own, cutaneous C. acnes strains in the pathogenesis of shoulder arthroplasty infection. Our findings support the importance of further research focused on perioperative decolonization and management of endogenous bacteria that are likely to be introduced into the arthroplasty wound at the time of skin incision.

Development of advanced control material for reverse transcription-mediated bacterial nucleic acid amplification tests.

Detection of bacterial RNA by nucleic acid amplification tests (NAATs), such as reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), offers distinct advantages over DNA-based methods. However, such assays also present challenges in ascertaining positive and internal control material that can reliably monitor success over all phases of testing (bacterial lysis, nucleic acid recovery, reverse transcription, amplification, and signal detection): since they are unable to distinguish between amplification of bacterial RNA transcripts and the DNA templates that encode them, using intact organisms as controls can inform cell lysis but not successful detection of RNA. We developed a control strategy for RNA-based bacterial NAATs that allows ready discrimination of RNA from DNA templates using self-splicing bacterial introns, such that those nucleic acids ultimately encode different sequences. We engineered two vectors encoding synthetic transgenes based on this principle, one that is active in the Gram-negative bacterium Escherichia coli and one that functions in both E. coli and the Gram-positive organism Staphylococcus aureus. We subsequently designed RT-LAMP assays that either target RNA and DNA from transgenic organisms or target RNA exclusively and demonstrated the specificity of amplification using purified nucleic acids. Using multiplex fluorescent RT-LAMP of heat-lysed specimens, we showed the practicality of deploying such transgenic organisms as an internal control to ascertain sample integrity and assay performance during clinical diagnostic testing. Our approach has broad utility for RNA-based bacterial NAATs, especially point-of-care assays and other applications where nucleic acids are nonspecifically liberated for testing.

Contribution of the patient microbiome to surgical site infection and antibiotic prophylaxis failure in spine surgery.

Despite modern antiseptic techniques, surgical site infection (SSI) remains a leading complication of surgery. However, the origins of SSI and the high rates of antimicrobial resistance observed in these infections are poorly understood. Using instrumented spine surgery as a model of clean (class I) skin incision, we prospectively sampled preoperative microbiomes and postoperative SSI isolates in a cohort of 204 patients. Combining multiple forms of genomic analysis, we correlated the identity, anatomic distribution, and antimicrobial resistance profiles of SSI pathogens with those of preoperative strains obtained from the patient skin microbiome. We found that 86% of SSIs, comprising a broad range of bacterial species, originated endogenously from preoperative strains, with no evidence of common source infection among a superset of 1610 patients. Most SSI isolates (59%) were resistant to the prophylactic antibiotic administered during surgery, and their resistance phenotypes correlated with the patient's preoperative resistome (P = 0.0002). These findings indicate the need for SSI prevention strategies tailored to the preoperative microbiome and resistome present in individual patients.

Evaluation of Pre-Analytical Variables for Human Papillomavirus Primary Screening from Self-Collected Vaginal Swabs.

Human papillomavirus (HPV) primary screening is an effective approach to assessing cervical cancer risk. Self-collected vaginal swabs can expand testing access, but the data defining analytical performance criteria necessary for adoption of self-collected specimens are limited, especially for those occurring outside the clinic, where the swab remains dry during transport. Here, we evaluated the performance of self-collected vaginal swabs for HPV detection using the Cobas 6800. There was insignificant variability between swabs self-collected by the same individual (n = 15 participants collecting 5 swabs per participant), measured by amplification of HPV and human ß-globin control DNA. Comparison of self-collected vaginal swab and provider-collected cervical samples (n = 144 pairs) proved highly concordant for HPV detection (total agreement = 90.3%; positive percentage agreement = 84.2%). There was no relationship between the number of dry storage days and amplification of HPV (n = 68; range, 4 to 41 days). Exposure of self-collected dry swabs to extreme summer and winter temperatures did not affect testing outcomes. A second internal control (RNase P) demonstrated that lack of amplification for ß-globin from self-collected specimens was consistent with poor, but not absent, cellularity. These data suggest that self-collected vaginal samples enable accurate clinical HPV testing, and that extended ambient dry storage or exposure to extreme temperatures does not influence HPV detection. Furthermore, lack of ß-globin amplification in HPV-negative samples accurately identified participants who required recollection.

Alterations in the fecal microbiota in patients with advanced cystic fibrosis liver disease after 6 months of elexacaftor/tezacaftor/ivacaftor.

BACKGROUND: Cystic fibrosis associated liver disease (CFLD) carries a significant disease burden with no effective preventive therapies. According to the gut-liver axis hypothesis for CFLD pathogenesis, dysbiosis and increased intestinal inflammation and permeability permit pathogenic bacterial translocation into the portal circulation, leading to hepatic inflammation and fibrosis. Evaluating the effect of CFTR (cystic fibrosis transmembrane conductance regulator) modulation with elexacaftor/tezacaftor/ivacaftor (ETI) may help determine the role of CFTR in CFLD and increase understanding of CFLD pathogenesis, which is critical for developing therapies. We aimed to characterize the fecal microbiota in participants with CF with and without advanced CFLD (aCFLD) before and after ETI. METHODS: This is an ancillary analysis of stool samples from participants ages ≥12 y/o enrolled in PROMISE (NCT04038047). Included participants had aCFLD (cirrhosis with or without portal hypertension, or non-cirrhotic portal hypertension) or CF without liver disease (CFnoLD). Fecal microbiota were defined by shotgun metagenomic sequencing at baseline and 1 and 6 months post-ETI. RESULTS: We analyzed 93 samples from 34 participants (11 aCFLD and 23 CFnoLD). Compared to CFnoLD, aCFLD had significantly higher baseline relative abundances of potential pathogens Streptococcus salivarius and Veillonella parvula. Four of 11 aCFLD participants had an initially abnormal fecal calprotectin that normalized 6 months post-ETI, correlating with a significant decrease in S. salivarius and a trend towards decreasing V. parvula. CONCLUSIONS: These results support an association between dysbiosis and intestinal inflammation in CFLD with improvements in both post-ETI, lending further support to the gut-liver axis in aCFLD.

Longitudinal profiling of the intestinal microbiome in children with cystic fibrosis treated with elexacaftor-tezacaftor-ivacaftor.

The intestinal microbiome influences growth and disease progression in children with cystic fibrosis (CF). Elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA), the newest pharmaceutical modulator for CF, restores the function of the pathogenic mutated CF transmembrane conductance regulator (CFTR) channel. We performed a single-center longitudinal analysis of the effect of ELX/TEZ/IVA on the intestinal microbiome, intestinal inflammation, and clinical parameters in children with CF. Following ELX/TEZ/IVA, children with CF had significant improvements in body mass index and percent predicted forced expiratory volume in one second, and required fewer antibiotics for respiratory infections. Intestinal microbiome diversity increased following ELX/TEZ/IVA coupled with a decrease in the intestinal carriage of Staphylococcus aureus, the predominant respiratory pathogen in children with CF. There was a reduced abundance of microbiome-encoded antibiotic resistance genes. Microbial pathways for aerobic respiration were reduced after ELX/TEZ/IVA. The abundance of microbial acid tolerance genes was reduced, indicating microbial adaptation to increased CFTR function. In all, this study represents the first comprehensive analysis of the intestinal microbiome in children with CF receiving ELX/TEZ/IVA.IMPORTANCECystic fibrosis (CF) is an autosomal recessive disease with significant gastrointestinal symptoms in addition to pulmonary complications. Recently approved treatments for CF, CF transmembrane conductance regulator (CFTR) modulators, are anticipated to substantially improve the care of people with CF and extend their lifespans. Prior work has shown that the intestinal microbiome correlates with health outcomes in CF, particularly in children. Here, we study the intestinal microbiome of children with CF before and after the CFTR modulator, ELX/TEZ/IVA. We identify promising improvements in microbiome diversity, reduced measures of intestinal inflammation, and reduced antibiotic resistance genes. We present specific bacterial taxa and protein groups which change following ELX/TEZ/IVA. These results will inform future mechanistic studies to understand the microbial improvements associated with CFTR modulator treatment. This study demonstrates how the microbiome can change in response to a targeted medication that corrects a genetic disease.

Evaluation of Fourier transform-infrared spectroscopy (FT-IR) as a control measure for nosocomial outbreak investigations.

Whole-genome sequencing (WGS) provides greater resolution than other molecular epidemiology strategies and is emerging as a new gold standard approach for microbial strain typing. The Bruker IR Biotyper is designed as a screening tool to identify bacterial isolates that require WGS to establish accurate relationships, but its performance and utility in nosocomial outbreak investigations have not been thoroughly investigated. Here, we evaluated the IR Biotyper by retrospectively examining isolates tested by WGS during investigations of potential nosocomial transmission events or outbreaks. Ninety-eight clinical isolates from 14 different outbreak investigations were examined: three collections of Acinetobacter baumannii (n = 2, n = 9, n = 5 isolates in each collection), one of Escherichia coli (n = 16), two of Pseudomonas aeruginosa (n = 2 and n = 5), two of Serratia marcescens (n = 9 and n = 7), five of Staphylococcus aureus (n = 8, n = 4, n = 3, n = 3, n = 17), and one of Stenotrophomonas maltophilia (n = 8). Linear regression demonstrated a weak, positive correlation between the number of pairwise genome-wide single-nucleotide polymorphisms (SNPs) and IR Biotyper spectral distance values for Gram-positive (r = 0.43, P ≤ 0.0001), Gram-negative (r = 0.1554, P = 0.0639), and all organisms combined (r = 0.342, P ≤ 0.0001). Overall, the IR Biotyper had a positive predictive value (PPV) of 55.81% for identifying strains that were closely related by genomic identity, but a negative predictive value (NPV) of 86.79% for identifying unrelated isolates. When experimentally adjusted cut-offs were applied to A. baumannii, P. aeruginosa, and E. coli, the PPV was 62% for identifying strains that were closely related and the NPV was 100% for identifying unrelated isolates. Implementation of the IR Biotyper as a screening tool in this cohort would have reduced the number of Gram-negative isolates requiring further WGS analysis by 50% and would reduce the number of S. aureus isolates needing WGS resolution by 48%.

Transposon sequencing identifies genes impacting Staphylococcus aureus invasion in a human macrophage model.

Staphylococcus aureus is a facultative intracellular pathogen in many host cell types, facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce highly saturated transposon mutant libraries in S. aureus and performed transposon insertion sequencing (Tn-Seq) to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 108 were identified as common across both S. aureus strains, with most (n = 106) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a subset of genes. Of the 20 genes passing validation, seven had previously identified roles in S. aureus virulence, and 13 were newly implicated. Validated genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of de novo mutations occurring in groups (n = 237) of clonally related S. aureus isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater in vivo purifying selection in conditionally essential candidate genes than those not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by S. aureus, highlights strain-specific differences in phenotypic effects of effector genes, and provides evidence for selection of candidate genes identified by Tn-Seq analyses during chronic airway infection in CF patients in vivo.

Longitudinal Profiling of the Intestinal Microbiome in Children with Cystic Fibrosis Treated with Elexacaftor-Tezacaftor-Ivacaftor.

The intestinal microbiome influences growth and disease progression in children with cystic fibrosis (CF). Elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA), the newest pharmaceutical modulator for CF, restores function of the pathogenic mutated CFTR channel. We performed a single-center longitudinal analysis of the effect of ELX/TEZ/IVA on the intestinal microbiome, intestinal inflammation, and clinical parameters in children with CF. Following ELX/TEZ/IVA, children with CF had significant improvements in BMI, ppFEV1 and required fewer antibiotics for respiratory infections. Intestinal microbiome diversity increased following ELX/TEZ/IVA coupled with a decrease in the intestinal carriage of Staphylococcus aureus, the predominant respiratory pathogen in children with CF. There was a reduced abundance of microbiome-encoded antibiotic-resistance genes. Microbial pathways for aerobic respiration were reduced after ELX/TEZ/IVA. The abundance of microbial acid tolerance genes was reduced, indicating microbial adaptation to increased CFTR function. In all, this study represents the first comprehensive analysis of the intestinal microbiome in children with CF receiving ELX/TEZ/IVA.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

In Silico Evaluation of Variant Calling Methods for Bacterial Whole-Genome Sequencing Assays.

Identification and analysis of clinically relevant strains of bacteria increasingly relies on whole-genome sequencing. The downstream bioinformatics steps necessary for calling variants from short-read sequences are well-established but seldom validated against haploid genomes. We devised an in silico workflow to introduce single nucleotide polymorphisms (SNP) and indels into bacterial reference genomes, and computationally generate sequencing reads based on the mutated genomes. We then applied the method to Mycobacterium tuberculosis H37Rv, Staphylococcus aureus NCTC 8325, and Klebsiella pneumoniae HS11286, and used the synthetic reads as truth sets for evaluating several popular variant callers. Insertions proved especially challenging for most variant callers to correctly identify, relative to deletions and single nucleotide polymorphisms. With adequate read depth, however, variant callers that use high quality soft-clipped reads and base mismatches to perform local realignment consistently had the highest precision and recall in identifying insertions and deletions ranging from1 to 50 bp. The remaining variant callers had lower recall values associated with identification of insertions greater than 20 bp.

Pulmonary Co-Infections Detected Premortem Underestimate Postmortem Findings in a COVID-19 Autopsy Case Series.

Bacterial and fungal co-infections are reported complications of coronavirus disease 2019 (COVID-19) in critically ill patients but may go unrecognized premortem due to diagnostic limitations. We compared the premortem with the postmortem detection of pulmonary co-infections in 55 fatal COVID-19 cases from March 2020 to March 2021. The concordance in the premortem versus the postmortem diagnoses and the pathogen identification were evaluated. Premortem pulmonary co-infections were extracted from medical charts while applying standard diagnostic definitions. Postmortem co-infection was defined by compatible lung histopathology with or without the detection of an organism in tissue by bacterial or fungal staining, or polymerase chain reaction (PCR) with broad-range bacterial and fungal primers. Pulmonary co-infection was detected premortem in significantly fewer cases (15/55, 27%) than were detected postmortem (36/55, 65%; p < 0.0001). Among cases in which co-infection was detected postmortem by histopathology, an organism was identified in 27/36 (75%) of cases. Pseudomonas, Enterobacterales, and Staphylococcus aureus were the most frequently identified bacteria both premortem and postmortem. Invasive pulmonary fungal infection was detected in five cases postmortem, but in no cases premortem. According to the univariate analyses, the patients with undiagnosed pulmonary co-infection had significantly shorter hospital (p = 0.0012) and intensive care unit (p = 0.0006) stays and significantly fewer extra-pulmonary infections (p = 0.0021). Bacterial and fungal pulmonary co-infection are under-recognized complications in critically ill patients with COVID-19.

MLH1 Loss in Primary Prostate Cancer.

PURPOSE: Among mismatch repair-deficient (MMRd) prostate cancers, loss of MLH1 is relatively uncommon and few cases have been reported in detail. METHODS: Here, we describe the molecular features of two cases of primary prostate cancer with MLH1 loss detected by immunohistochemistry, and in one case, confirmed via transcriptomic profiling. RESULTS: Both cases were microsatellite stable on standard polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing, but showed evidence of MSI on a newer PCR-based long mononucleotide repeat (LMR) assay and by next-generation sequencing. Germline testing was negative for Lynch syndrome-associated mutations in both cases. Targeted or whole-exome tumor sequencing using multiple commercial/academic platforms (Foundation, Tempus, JHU, and UW-OncoPlex) showed modestly elevated, though variable, tumor mutation burden estimates (2.3-10 mutations/Mb) consistent with MMRd, but without identifiable pathogenic single-nucleotide or indel mutations in MLH1. Copy-number analysis confirmed biallelic MLH1 loss in one case and monoallelic MLH1 loss in the second case, without evidence of MLH1 promoter hypermethylation in either. The second patient was treated with single-agent pembrolizumab and demonstrated a short-lived prostate-specific antigen response. CONCLUSION: These cases highlight the challenges in identifying MLH1-deficient prostate cancers using standard MSI testing and commercial sequencing panels, and support the utility of immunohistochemical assays and LMR- or sequencing-based MSI testing for detection of MMRd prostate cancers.