Re-examining the classification of abdominal pseudocysts as surgical site infections through review of broad-range polymerase chain reaction results.

BACKGROUND: The Hydrocephalus Clinical Research Network-quality group (HCRNq) historically defined all abdominal pseudocysts associated with a ventriculoperitoneal shunt as a surgical site infection regardless of culture result. METHODS: We retrospectively reviewed broad-range polymerase chain reaction (BRPCR) results sent between January 2017 and July 2023 from abdominal pseudocyst fluid sent from hospitals around the country to a reference laboratory to help further characterize these collections. RESULTS: A total of 19 samples were tested via BRPCR between 1/2017 and 7/2023. Two (10.5 %) had organisms identified; one with Staphylococcus epidermidis and one with Candida parapsilosis. No fastidious organisms that would be expected to not grow with typical culture techniques were identified. CONCLUSIONS: Few abdominal pseudocysts had organisms identified by BRPCR, suggesting that not all pseudocysts are due to infectious causes. Consideration should be given to alternate causes of pseudocyst development when cultures are negative.

A retrospective observational study of mNGS test utilization to examine the role of diagnostic stewardship at two academic medical centers.

Given the cost and unclear clinical impact of metagenomic next-generation sequencing (mNGS), laboratory stewardship may improve utilization. This retrospective observational study examines mNGS results from two academic medical centers employing different stewardship approaches. Eighty mNGS orders [54 cerebrospinal fluid (CSF) and 26 plasma] were identified from 2019 to 2021 at the University of Washington (UW), which requires director-level approval for mNGS orders, and the University of Utah (Utah), which does not restrict ordering. The impact of mNGS results and the relationship to traditional microbiology orders were evaluated. Nineteen percent (10/54) of CSF and 65% (17/26) of plasma studies detected at least one organism. Compared to CSF results, plasma results more frequently identified clinically significant organisms (31% vs 7%) and pathogens not detected by traditional methods (12% vs 0%). Antibiotic management was more frequently impacted by plasma versus CSF results (31% vs 4%). These outcome measures were not statistically different between study sites. The number and cumulative cost of traditional microbiology tests at UW were greater than Utah for CSF mNGS testing (UW: 46 tests, $6,237; Utah: 26 tests, $2,812; P < 0.05) but similar for plasma mNGS (UW: 31 tests, $3,975; Utah: 21 tests, $2,715; P = 0.14). mNGS testing accounted for 30%-50% of the total microbiology costs. Improving the diagnostic performance of mNGS by stewardship remains challenging due to low positivity rates and difficulties assessing clinical impact. From a fiscal perspective, stewardship efforts should focus on reducing testing in low-yield populations given the high costs of mNGS relative to overall microbiology testing expenditures. IMPORTANCE: Metagenomic next-generation sequencing (mNGS) stewardship practices remain poorly standardized. This study aims to provide actionable insights for institutions that seek to reduce the unnecessary usage of mNGS. Importantly, we highlight that clinical impact remains challenging to measure without standardized guidelines, and we provide an actual cost estimate of microbiology expenditures on individuals undergoing mNGS.

Histologic Features of Mycobacterial Spindle Cell Pseudotumors: A Multi-institutional Clinicopathologic Analysis of 14 Cases.

Mycobacterial spindle cell pseudotumors (MSPs) are a rare and diagnostically challenging manifestation of non-tuberculous mycobacterial (NTM) infections. Proper recognition of these pseudotumors is important because they are treatable and benign. In this study, we evaluated the morphologic patterns of MSPs to improve their pathologic identification. Clinical and morphologic features of 14 MSPs were analyzed. Histologic factors evaluated included the architectural growth pattern of spindled or epithelioid macrophages, granulomas and their location within the lesion, neutrophilic microabscesses, multinucleated giant cells, necrosis, and effacement of background tissue. The composition of inflammatory infiltrates, organism density by acid-fast staining, and stromal changes were also assessed. In addition, 8 of 14 cases underwent molecular microbiology identification by a clinical amplicon-sequencing assay for non-tuberculous mycobacteria. MSP sites included 2 bowel, 10 lymph nodes, 1 liver, and 1 extremity. Cases with available clinical history (n=10) all occurred in immunocompromised patients. All demonstrated effacement of normal structures with spindled cells arranged in a storiform or fascicular architectural pattern. In addition, all cases showed lymphocytic inflammation, with prominent concurrent neutrophilic inflammation in 50% (7/14) of cases. Other morphologic findings included foamy histiocytes (64%, 9/14), peripherally situated granulomas (21%, 3/14), and neutrophilic microabscesses (21%, 3/14). All tested cases were positive for NTM by PCR methods. Mycobacterium avium was the most commonly isolated pathogen (6/8). Mycobacterial spindle cell pseudotumors show predominantly spindled morphology that may be mistaken as a neoplasm. Surgical pathologists who evaluate lymph nodes, soft tissue, and gastrointestinal tissues should be aware of this spindled tumefactive phenomenon in the setting of immunocompromised patients. Recognition of key morphologic features of neutrophilic inflammation, peripheral granulomas, or foamy histiocytes within a spindled lesion can help guide the pathologist to a correct diagnosis of an inflammatory process secondary to infection rather than a spindle cell neoplasm. Accurate diagnosis to facilitate appropriate antimicrobial and/or surgical therapy requires a comprehensive evaluation combining clinical, histopathologic, and microbiological findings.

Risk factors and provider awareness of sexually transmitted enteric pathogens among men who have sex with men.

Sexual transmission of enteric pathogens among men who have sex with men (MSM) is well documented, although whether providers are cognizant of this risk when MSM patients present with gastrointestinal symptoms has not been studied. Over 34 months at a major tertiary metropolitan medical system, this study retrospectively analyzed 436 BioFire FilmArray Gastrointestinal results from 361 patients documented as MSM. An extensive chart review was performed, including specific sexual behaviors, socioeconomic risk factors, and whether providers charted a sexual history when a patient presented for care. Overall BioFire positivity rate was 62% with no significant difference in positivity between persons living with HIV and those without. Patients charted as sexually active had a significantly increased odds ratio (OR) of a positive result compared to those who were not. Anilingus had the highest OR. Providers charted any type of sexual history in 40.6% of cases, and HIV/infectious disease providers were significantly more likely to do this compared to other subspecialties. Sexual transmission of enteric pathogens within MSM is ongoing, and patients are at risk regardless of living with HIV. Not all sexual behaviors have the same associated risk, highlighting opportunities to decrease transmission. Increased provider vigilance and better patient education on sexual transmission of enteric pathogens are needed to reduce the disease burden. IMPORTANCE: Our work adds several key findings to the growing body of literature describing the epidemiology of enteric pathogens as sexually transmitted infections among men who have sex with men (MSM). We analyzed clinical test results, housing status, provider awareness, sexual behaviors, and symptoms for 361 patients. We found that any sexual activity was associated with an increased risk of diarrheal pathogen detection, whereas being unhoused was not a risk factor. These findings suggest separate transmission networks between unhoused persons, who are also at risk of infectious diarrhea, and MSM. Moreover, our study suggested low awareness among patient-facing clinicians that diarrheal pathogens can be sexually transmitted. Together, our findings indicate an important opportunity to disrupt transmission cycles by educating clinicians on how to assess and counsel MSM patients.

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%.

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.

An Unnamed Human Oral Bergeyella sp. as the Cause of an Unusual Bacterial Keratitis.

Purpose. We report a case of bacterial keratitis secondary to an undescribed Bergeyella sp. Bergeyella spp. are not easily cultured, and many reports have identified unculturable isolates through broad-range bacterial polymerase chain reaction (PCR). Observations. A healthy 29-year-old male was attempting to repair an acrylic cannabis water pipe when it shattered and a fragment hit him in the left eye. Two weeks later, he presented with foreign body sensation, scleral injection, and photophobia that were refractory to prolonged corticosteroid therapy. Following a subconjunctival triamcinolone injection, the patient developed a hypopyon and multifocal, midstromal, epithelized corneal infiltrates. Broad-range PCR of the aqueous fluid detected deoxyribonucleic acid closely matching the Bergeyella genus. Empiric treatment directed toward gram-negative bacteria led to the clinical resolution of the inflammation. Conclusions and Importance. This is the first reported case of ocular inflammation secondary to a Bergeyella spp.. As broad-range PCR testing becomes more accessible, we anticipate that additional PCR-positive and culture-negative scenarios will occur.

Programmable peroxidase-assisted signal amplification enables flexible detection of nucleic acid targets in cellular and histopathological specimens.

In situ hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate co-visualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid, and compatible with a broad range of optical systems. Here, we introduce a unified technical platform, termed 'pSABER', for the amplification of ISH signals in cell and tissue systems. pSABER decorates the in situ target with concatemeric binding sites for a horseradish peroxidase-conjugated oligonucleotide which can then catalyze the massive localized deposition of fluorescent or colorimetric substrates. We demonstrate that pSABER effectively labels DNA and RNA targets, works robustly in cultured cells and challenging formalin fixed paraffin embedded (FFPE) specimens. Furthermore, pSABER can achieve 25-fold signal amplification over conventional signal amplification by exchange reaction (SABER) and can be serially multiplexed using solution exchange. Therefore, by linking nucleic acid detection to robust signal amplification capable of diverse readouts, pSABER will have broad utility in research and clinical settings.

Bartonella spp. Infections Identified by Molecular Methods, United States.

Molecular methods can enable rapid identification of Bartonella spp. infections, which are difficult to diagnose by using culture or serology. We analyzed clinical test results of PCR that targeted bacterial 16S rRNA hypervariable V1-V2 regions only or in parallel with PCR of Bartonella-specific ribC gene. We identified 430 clinical specimens infected with Bartonella spp. from 420 patients in the United States. Median patient age was 37 (range 1-79) years; 62% were male. We identified B. henselae in 77%, B. quintana in 13%, B. clarridgeiae in 1%, B. vinsonii in 1%, and B. washoensis in 1% of specimens. B. quintana was detected in 83% of cardiac specimens; B. henselae was detected in 34% of lymph node specimens. We detected novel or uncommon Bartonella spp. in 9 patients. Molecular diagnostic testing can identify Bartonella spp. infections, including uncommon and undescribed species, and might be particularly useful for patients who have culture-negative endocarditis or lymphadenitis.

Diagnosis, Prognosticators, and Management of Acute Invasive Fungal Rhinosinusitis: Multidisciplinary Consensus Statement and Evidence-Based Review with Recommendations.

BACKGROUND: Acute invasive fungal sinusitis (AIFS) is an aggressive disease that requires prompt diagnosis and multidisciplinary treatment given its rapid progression. However, there is currently no consensus on diagnosis, prognosis, and management strategies for AIFS, with multiple modalities routinely employed. The purpose of this multi-institutional and multidisciplinary evidence-based review with recommendations (EBRR) is to thoroughly review the literature on AIFS, summarize the existing evidence, and provide recommendations on the management of AIFS. METHODS: The PubMed, EMBASE, and Cochrane databases were systematically reviewed from inception through January 2022. Studies evaluating management for orbital, non-sinonasal head and neck, and intracranial manifestations of AIFS were included. An iterative review process was utilized in accordance with EBRR guidelines. Levels of evidence and recommendations on management principles for AIFS were generated. RESULTS: A review and evaluation of published literature was performed on 12 topics surrounding AIFS (signs and symptoms, laboratory and microbiology diagnostics, endoscopy, imaging, pathology, surgery, medical therapy, management of extrasinus extension, reversing immunosuppression, and outcomes and survival). The aggregate quality of evidence was varied across reviewed domains. CONCLUSION: Based on the currently available evidence, judicious utilization of a combination of history and physical examination, laboratory and histopathologic techniques, and endoscopy provide the cornerstone for accurate diagnosis of AIFS. In addition, AIFS is optimally managed by a multidisciplinary team via a combination of surgery (including resection whenever possible), antifungal therapy, and correcting sources of immunosuppression. Higher quality (i.e., prospective) studies are needed to better define the roles of each modality and determine diagnosis and treatment algorithms.

Relapsing Fever Caused by Borrelia lonestari after Tick Bite in Alabama, USA.

We report an immunocompromised patient in Alabama, USA, 75 years of age, with relapsing fevers and pancytopenia who had spirochetemia after a tick bite. We identified Borrelia lonestari by using PCR, sequencing, and phylogenetic analysis. Increasing clinical availability of molecular diagnostics might identify B. lonestari as an emerging tickborne pathogen.

Variant APOL1 protein in plasma associates with larger particles in humans and mouse models of kidney injury.

BACKGROUND: Genetic variants in apolipoprotein L1 (APOL1), a protein that protects humans from infection with African trypanosomes, explain a substantial proportion of the excess risk of chronic kidney disease affecting individuals with sub-Saharan ancestry. The mechanisms by which risk variants damage kidney cells remain incompletely understood. In preclinical models, APOL1 expressed in podocytes can lead to significant kidney injury. In humans, studies in kidney transplant suggest that the effects of APOL1 variants are predominantly driven by donor genotype. Less attention has been paid to a possible role for circulating APOL1 in kidney injury. METHODS: Using liquid chromatography-tandem mass spectrometry, the concentrations of APOL1 were measured in plasma and urine from participants in the Seattle Kidney Study. Asymmetric flow field-flow fractionation was used to evaluate the size of APOL1-containing lipoprotein particles in plasma. Transgenic mice that express wild-type or risk variant APOL1 from an albumin promoter were treated to cause kidney injury and evaluated for renal disease and pathology. RESULTS: In human participants, urine concentrations of APOL1 were correlated with plasma concentrations and reduced kidney function. Risk variant APOL1 was enriched in larger particles. In mice, circulating risk variant APOL1-G1 promoted kidney damage and reduced podocyte density without renal expression of APOL1. CONCLUSIONS: These results suggest that plasma APOL1 is dynamic and contributes to the progression of kidney disease in humans, which may have implications for treatment of APOL1-associated kidney disease and for kidney transplantation.

Cokeromyces recurvatus Incidentally Found in a Patient with Gastric Outlet Obstruction.

Isolation of Cokeromyces recurvatus, a dimorphic mucormycete fungus, from clinical specimens poses a diagnostic challenge to physicians and laboratorians as this organism may represent a rare colonizer or true pathogen. Here, we report a case of Cokeromyces recurvatus present in a circumferential duodenal lesion. The patient is a 64-year-old with no past medical history, admitted with a three-week history of left upper quadrant abdominal pain. Computerized tomography scan identified duodenitis with significant gastric outlet obstruction, confirmed by the presence of a partially obstructing non-bleeding duodenal ulcer on upper endoscopy. Histology showed variably sized spherical structures without nuclei, reproductive tracts, or alimentary tracts. Small, clustered spherules representing putative endospores were observed within the larger structures and in the exudate. Based on the histology, the differential included Coccidioides spp, Emmonsia spp, or Chrysosporium spp. Additionally, gastric biopsies revealed concurrent Helicobacter pylori gastritis. The fungus was identified as C. recurvatus by broad-range fungal polymerase chain reaction performed on formalin-fixed paraffin-embedded biopsy tissue, as well as morphology and DNA sequencing of the cultured isolate. The fungus had low MICs to all major antifungal classes; however, in the context of the Helicobacter pylori infection, the patient was only treated with amoxicillin and clarithromycin with improvement in his symptoms before hospital discharge. Only three cases of Cokeromyces recurvatus isolated from the GI tract have been reported; this case highlights a unique clinical presentation in the small bowel in a patient without underlying medical conditions.

The transfusion service laboratory in virtual reality.

Due to the global SARS-CoV-2 pandemic, in-person laboratory medicine clerkships were converted to distance learning. The remote clerkship format provided advantages of allowing participation of students from more locations and greater scheduling flexibility but provided new challenges of maintaining learner engagement and providing experiential content of the laboratory environment. Gamification of educational content is one educational modality that has shown effectiveness in a multitude of different contexts to increase learner engagement and retention. Therefore, we created an interactive, educational 360° virtual reality walkthrough tour using off-the-shelf commercially available 360° cameras and software of the Transfusion service and Microbiology Laboratories. The process consists of taking multiple 360° still-images within the space, color-correction, blurring the faces of staff or sensitive information, adding navigation buttons, and other interactive elements. The virtual tours were used for both recruitment and education with further plans to integrate the learning modality into the curriculum. The clerkship is likely to remain as partially or fully as remote learning so such walkthrough tours will continue to remain relevant. This technology can be applied globally to other departments and institutions for education or recruitment.

High Clinical Impact of Broad-Range Fungal PCR in Suspected Fungal Sinusitis.

Broad-range fungal PCR is a powerful tool for identifying pathogens directly from patient specimens; however, reported estimates of clinical utility vary and costs discourage universal testing. We investigated the diagnostic and clinical utility of broad-range fungal PCR by examining 9 years of results from sinonasal specimens, hypothesizing that this anatomic location would identify immunocompromised patients at high risk for invasive fungal disease. We retrospectively identified 644 PCRs and 1,446 fungal cultures from sinus sites. To determine the relative performance of each testing modality, we performed chart review on 52 patients having specimens submitted for culture and PCR on the same day. Positivity rates were significantly higher for PCR (37.1%) than culture (13.7%) but similar for formalin-fixed and fresh tissues (42.3% versus 34.6%). Relative to culture, PCR had significantly faster turnaround time to both preliminary (94.5 versus 108.8 h) and final positive (137.9 versus 278.5 h) results. Among chart-reviewed patients, 88% were immunocompromised, 65% had proven or probable fungal disease, and testing sensitivities for culture and PCR (67.5% and 85.0%) were not statistically different. Nevertheless, PCR identified pathogens not recovered by culture in 14.9% of cases and informed clinical decision-making in 16.7% of all reviewed cases, and sensitivity of PCR combined with culture (90.0%) was higher than that of culture alone. We conclude that broad-range fungal PCR is frequently informative for patients at risk of serious fungal disease and is complementary to and has faster turnaround time than culture. Formalin-fixed tissue does not adversely affect diagnostic yield, but anatomic site may impact assay positivity rates.