Diagnostic pitfalls in effusion fluid cytology

Effusion fluid cytology has propensity for both false positives (in up to 0.5%) and false negatives (in up to 30%) results. Methodical approach from collection step to final interpretation stage could prevent both false positives and false negatives, if the interpreter is familiar with various factors responsible for diagnostic pitfalls in effusion fluid cytology. For this discussion, these factors are categorized as mentioned below: Surface tension-related alterations in cytomorphology Improper specimen processing Many faces of reactive mesothelial cells, overlapping with those of cancer cells Proliferation-related features Degenerative changes, such as nuclear hyperchromasia and cytoplasmic vacuolation Unexpected patterns and unusual entities.

799. A Pseudo-Outbreak of Pseudomonas fluorescens Infections

Background Pseudomonas fluorescens is a water-borne pathogen that has been associated with outbreaks from transfusion of contaminated blood products or medical equipment. Our institution had a cluster of cultures that grew an uncommonly encountered microbe P. fluorescens within a period of one week. This prompted an internal investigation. We summarize the investigational process that led to the resolution of this pseudo-outbreak. Methods We conducted a retrospective chart review of surgical and non-surgical patients with cultures positive for P. fluorescens from July 2nd to July 8th 2020. Baseline patient characteristics, clinical course, laboratory data, use of blood-associated products, and microbiology cultures were analyzed. Results Eight patients were identified with positive tissue cultures for P. fluorescens. Among those, 5 specimens (62.5%) were from osteoarticular sites (1 prosthetic hip, 1 prosthetic knee, 1 right foot, 1 sternum, and 1 vertebral source). One culture (12.5%) was obtained from a sacral soft tissue wound. Two tissue specimens (25%) were collected from respiratory sites (1 lung tissue and 1 bronchoalveolar lavage). No association with specific surgical personnel or operating room was identified. During routine specimen processing, a small amount of sterile normal saline is added to the conical grinder prior to culture preparation. It was discovered that a non-sterile normal saline had been inadvertently utilized during that step. These eight tissue specimens were subsequently reprocessed with sterile solution; P. fluorescens was not re-isolated. Specimen processing protocols were reinforced. Adjustment of antimicrobial therapy was made accordingly without reported subsequent adverse clinical outcomes. Conclusion A multi-faceted team approach in collaboration with Infection Prevention, Infectious Diseases, Surgery, operating room personnel, and Microbiology identified an unintended breakdown in sterile laboratory protocols which resulted in a cluster of falsely positive cultures. An increased incidence of infection with an uncommon pathogen initiated a prompt investigation that resulted in the identification of a pseudo-outbreak event. Disclosures All Authors: No reported disclosures

Influence of apical preparation size and final irrigation protocol on the debridement of oval root canals

This study assessed the influence of apical preparation size and final irrigation protocol on the debridement of the apical third of oval root canals of mandibular molars. Seventy-seven distal roots were divided into 7 groups (n = 11): Control: without instrumentation or irrigation; Group 30CI: ProTaper Next (up to size 30; PTN) + conventional irrigation (CI); Group 30UAI: PTN + ultrasonically activated irrigation (UAI); Group 30XPF: PTN + XP-endo Finisher (XPF); Group 40CI: PTN + ProDesign Logic (up to size 40; PDL) + CI; Group 40UAI: PTN + PDL + UAI; and Group 40XPF: PTN + PDL + XPF. The total volume of irrigating solutions used per root canal in all the experimental groups was 33 mL of 2.5% sodium hypochlorite (NaOCl) and 6 mL of 17% ethylenediaminetetraacetic acid (EDTA). After specimen processing and histological analysis under a digital microscope (100x), the percentages of untouched canal walls (UCW) and remaining debris (RD) were assessed using Image J software. A descriptive and exploratory analysis was conducted, indicating that the data failed to meet the assumptions of an analysis of variance. Therefore, generalized linear models were used to assess the effects of the different instrumentation and irrigation protocols, as well as the interaction among them, on the percentage of UCW and RD. No significant difference was found among the irrigation protocols regarding the percentage of UCW, irrespective of apical preparation size (p>0.05). However, UCW and RD were significantly lower in groups 40CI, 40UAI and 40XPF than in groups 30CI, 30UAI and 30XPF (p<0.05). The percentage of RD was significantly lower in the UAI and XPF groups than in the CI groups, irrespective of apical preparation size (p<0.05). The difference between preparation sizes 30 and 40, with respect to RD, was higher when CI was used (p<0.05). In conclusion, instrumentation up to apical preparation size 40 resulted in lower percentages of UCW and RD than up to apical preparation size 30. Use of UAI or XPF resulted in lower percentages of RD than CI.

Xpert MTB/RIF Ultra is highly sensitive for the diagnosis of tuberculosis lymphadenitis in an HIV-endemic setting

Background: Tuberculosis lymphadenitis (TBL) is the most common extrapulmonary TB (EPTB) manifestation. Xpert MTB/RIF Ultra (Ultra) is a World Health Organization-endorsed diagnostic test, but performance data for TBL, including on non-invasive specimens, are limited. Methods: Fine needle aspiration biopsies (FNABs) from outpatients (≥18 years) with presumptive TBL (n=135) underwent: 1) routine Xpert (later Ultra once programmatically available), 2) a MGIT 960 culture (if Xpert- or Ultra-negative, or rifampicin-resistant), and 3) study Ultra. Concentrated paired urine underwent Ultra. Primary analyses used a microbiological reference standard (MRS). Results: In a head-to-head comparison (n=92) of FNAB study Ultra and Xpert, Ultra had increased sensitivity [91% (95% confidence interval 79, 98) vs. 72% (57, 84); p=0.016] and decreased specificity [76% (61, 87) vs. 93% (82, 99); p=0.020], and detected patients not on treatment. HIV nor alternative reference standards affected sensitivity and specificity. In patients with both routine and study Ultras, the latter detected more cases [+20% (0, 42); p=0.034] and, further indicative of potential laboratory-based room-for-improvement (e.g., specimen processing optimisation), false-negative study Ultras were more inhibited than true-positives. Study Ultra false-positives had less mycobacterial DNA than true-positives [trace-positive proportions 59% (13/22) vs. 12% (5/51); p<0.001]. “Trace” exclusion or recategorization removed potential benefits offered over Xpert. Urine Ultra had low sensitivity [18% (7, 35)]. Conclusions: Ultra on FNABs is highly sensitive and detects more TBL than Xpert. Patients with FNAB Ultra-positive “trace” results, most of whom will be culture-negative, may require additional clinical investigation. Urine Ultra could reduce the number of patients needing invasive sampling.

Challenges in Pathology Specimen Processing in the New Era of Precision Medicine

Context.— Precision therapies for patients with driver mutations can offer deep and durable responses that correlate with diagnosis, metastasis prognosis, and improvement in survival. Such targeted therapies will continue to increase, pushing us to change our traditional approaches. We needed to search for new tools to effectively integrate technological advancements into our practices because of their capability to improve the efficiency and accuracy of our diagnostic and treatment approaches. Perhaps nothing is as relevant as identifying and implementing new workflows for processing pathologic specimens and for improving communication of critical laboratory information to and from clinicians for appropriate care of patients in an efficient and timely manner. Objectives.— To define the gold standard in delivering the best care for patients, to identify gaps in the process, and to identify potential solutions that would improve our process, including gaps related to knowledge, skills, attitudes, and practices. Design.— We assembled a team across disciplines to systematically perform a gap analysis study to clarify the discrepancy between the current reality in pathology specimen processing and the desired optimal situation to deliver the results intended for patient care. Results.— A practical collaborative workflow for specimen management seeking the cooperation of the stakeholders in each medical discipline to provide guidelines in specimen collection, delivery, processing, and reporting of results with the ultimate goal of improving patients' outcomes is provided. Conclusions.— New tools are required to effectively integrate data-driven approaches in specimen processing to meet the new demands.

Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor

Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis (pyrazinamide-resistant organisms), may also be involved. Thus, the ability to rapidly detect and identify MTB from other MTBC members (e.g., M. bovis, Mycobacterium microti, Mycobacterium africanum) is essential for the prevention and treatment of TB. A novel diagnostic method for the rapid detection and differentiation of MTB, which employs multiplex loop-mediated isothermal amplification (mLAMP) combined with a nanoparticle-based lateral flow biosensor (LFB), was established (mLAMP-LFB). Two sets of specific primers that target the IS6110 and mtp40 genes were designed according to the principle of LAMP. Various pathogens were used to optimize and evaluate the mLAMP-LFB assay. The optimal conditions for mLAMP-LFB were determined to be 66°C and 40 min, and the amplicons were directly verified by observing the test lines on the biosensor. The LAMP assay limit of detection (LoD) was 125 fg per vessel for the pure genomic DNA of MTB and 4.8 × 103 CFU/ml for the sputum samples, and the analytical specificity was 100%. In addition, the whole process, including the clinical specimen processing (35 min), isothermal amplification (40 min), and result confirmation (1–2 min), could be completed in approximately 80 min. Thus, mLAMP-LFB is a rapid, reliable, and sensitive method that is able to detect representative members of MTBC and simultaneously differentiate MTB from other MTBC members, and it can be used as a potential screening tool for TB in clinical, field, and basic laboratory settings.

Standard PBMC cryopreservation selectively decreases detection of nine clinically-relevant T-cell markers

Biobanking is an operational component of various epidemiological studies and clinical trials. Although peripheral blood is routinely acquired and stored in biobanks, the effects of specimen processing on cell composition and clinically-relevant functional markers of T cells still require a systematic evaluation. Here we assessed 25 relevant T-cell markers and showed that the detection of nine membrane markers, e.g., PD-1, CTLA4, KLRG1, CD25, CD122, CD127 and others reflecting exhaustion, senescence and other functions was reduced among at least one T-cell subset following standard processing, although the frequency of CD4, CD8 and regulatory T cells was unaffected. Nevertheless, a six-month-long cryopreservation did not impair the expression levels of many other membrane and all the eight tested intracellular lineage or functional T-cell markers. Our findings uncover that several clinically-relevant markers are particularly affected by processing and the interpretation of those results in clinical trials and translational research should be done with caution.