Pritt, Bobbi S.

Abstract Effective filtration and concentration of stool specimens is an essential pre-analytical step for reducing fecal debris and improving organism recovery using microscopy-based ova and parasite (O&P) examination. This study evaluated three commercially available fecal sedimentation-based filtration/concentration systems – ParaPak SpinCon® (Meridian Bioscience), Mini Parasep SF® (Apacor), and the newly-available Paradevice® (Reingenuity) – for qualitative parasite detection and workflow logistics using conventional and artificial intelligence (AI)-assisted microscopy. Forty clinical stool specimens (20 parasite-positive and 20 parasite-negative) were processed with the 3 devices, and the resultant 120 wet mount and 120 trichrome-stained smear preparations were examined using conventional microscopy. Trichrome-stained slides were also scanned at 40x magnification using a Hamamatsu® NanoZoomerS360® flatbed digital slide scanner and images were analyzed using the Techcyte® Fusion Human Fecal Trichrome AI algorithm. Positive and indeterminate digital findings were confirmed by conventional glass slide microscopy. Slides and digital images were reviewed in a blinded manner. Concordance was assessed among the 360 initial evaluations (microscopy and AI-assisted), and discrepant parasitology results were resolved through re-review and specimen reprocessing as needed. Final qualitative agreement across slide/image evaluations using all three concentration systems was 100%. Minor discrepancies in protozoan and white/red blood cell detection/identification were noted in 5 and 7 cases, respectively, and likely reflected sampling and observer variability. While the three concentration systems produced equivalent qualitative results, the Paradevice® and Mini Parasep SF® offered the most streamlined workflows. These findings support the Paradevice® and Mini Parasep SF® as efficient, analytically equivalent systems that are compatible with traditional and AI-assisted O&P workflows. Importance Microscopy-based examination of stool specimens remains an important diagnostic tool for intestinal parasitic infections. Filtration and concentration methods improve test performance by increasing organism recovery and reducing obscuring fecal debris prior to evaluation. These methods may also facilitate the preparation of thin monolayer trichrome-stained slides for digital scanning and automated parasite detection. Because many clinical laboratories rely on commercial filtration and concentration systems, supply chain disruptions, product shortages, and manufacturer recalls threaten testing continuity, underscoring the need for validated alternatives. This study demonstrates that multiple commercially available concentration systems provide equivalent parasite detection while supporting both conventional microscopy and emerging digital workflows. These findings provide laboratories with evidence-based alternatives that enhance operational resilience without compromising diagnostic performance.

Abstract Background Babesia and Plasmodium are intraerythrocytic parasites that cause babesiosis and malaria, respectively. Significant associations between human blood group antigens and malaria are known; however, few studies have examined the relationship between blood group antigens and babesiosis. Study Design and Methods From June to August 2025, 100 Babesia polymerase chain reaction (PCR)− and 100 B. microti PCR+ human patient samples tested at a national reference laboratory underwent ABO and D phenotyping and red blood cell (RBC) genotyping of 37 antigens. Results The predicted phenotype of S‐s+ was found in 32% and 50% of Babesia − and B. microti + patients, respectively (χ 2 = 5.97, p = .015, odds ratio = 2.125, 95% confidence interval 1.18–3.78). Two B. microti + patients had the predicted phenotype of Fy(a‐b‐), meaning that Duffy is unlikely to be an essential invasion receptor. There were no statistically significant differences in ABO or D expression between the groups. Discussion Because multiple testing corrections were not applied to the statistical analyses, the S/s predicted phenotype results must be viewed as hypothesis‐generating. This may be the first study to link B. microti infection to human RBC S/s antigens, which are known to impact B. divergens and P. falciparum . Additional studies are needed to understand the impact of the babesiosis patient's blood type on B. microti infection and whether the transfusion of RBCs expressing specific antigens can impact the disease.

ABSTRACT Current laboratory testing for Lyme disease (LD) relies on serology. We evaluated the performance of standard two-tiered testing (STTT) and modified two-tiered testing (MTTT) algorithms using samples obtained from well-characterized patients with early LD in the U.S. East Coast and Upper Midwest. Participants with signs and symptoms of early LD (cases) and controls were enrolled by Lyme Disease Biobank. We compared the performance of four FDA-cleared STTT or MTTT algorithms using serum samples from 251 participants (107 cases, 69 with a convalescent draw; 144 endemic controls). At the initial blood draw, algorithm sensitivity ranged from 22% to 36%, with specificity ranging from 98% to 100%. MTTT algorithms showed higher sensitivity compared with STTT algorithms ( P ≤ 0.05). One STTT algorithm was less sensitive than the other ( P = 0.035), and there was no significant difference in sensitivity between MTTT algorithms. There was also discordance between algorithms; only 22 of the 45 samples classified as laboratory confirmed by Lyme Disease Biobank testing were positive using all algorithms evaluated. Likelihood of positive two-tiered serology among cases with a suspected erythema migrans (EM) skin lesion increased with longer lesion duration and/or when presenting with >1 constitutional symptom. Participants with suspected EM without constitutional symptoms were unlikely to test positive by any algorithm evaluated. Testing convalescent samples did not improve LD detection, and seroconversion was rare. While MTTT confirmed more early LD cases than STTT, all two-tiered algorithms evaluated were insensitive in this population. Novel diagnostics that improve laboratory confirmation for early LD are urgently needed. IMPORTANCE This study confirms that two-tiered serologic testing algorithms are insensitive in early Lyme disease, particularly within the first 2 weeks after symptom onset or when erythema migrans is not accompanied by constitutional symptoms. It also demonstrates that seroconversion is rare after antibiotic treatment. These results highlight the need for novel diagnostics for early Lyme disease that do not rely on serologic testing.

Abstract Background Leishmaniasis is a rare protozoan parasitic disease transmitted through the bite of infected Phlebotomus and Lutzomyia sandflies. Clinical presentation is variable due to numerous species and host immunity differences. We present our clinical experiences with CL to guide timely diagnosis and treatment in returning travelers. Methods We retrospectively analyzed a cohort of adult patients 18+ years old who were diagnosed with CL from 2005 and 2025. We describe the demographic, clinical, and diagnostic features of CL. Results Biopsy-confirmed CL was diagnosed in 20 returning travelers (Table 1) from various endemic regions, most commonly Costa Rica (Fig. 1). The median time from travel to symptom onset was 36.5 days (SD 102), with an additional 75.5 days (SD 87) prior to biopsy diagnosis. All patients were evaluated by dermatology and 70% were evaluated by infectious disease (n=14). On initial presentation, lesions were described as ulcerated (50%, n=10), nodular (30%, n=6), painful (20%, n=4), and necrotic (10%, n=2; Fig. 2). Most lesions were located on the upper extremity (55%, n=11), followed by the face (35%, n=7), lower extremity (30%, n=6), and trunk (15%, n=3). 15% of individuals (n=3) had constitutional symptoms. No patients had definitive mucosal involvement, however, 10% (n=2) endorsed lip edema. No patients had visceral involvement. Species level identification occurred in 14 cases (70%), of which the most common was L.V. panamensis (50%), L.L. mexicana (21%), L. V. guyanensis (14%), L. L. tropica (7%), and L. V. braziliensis (7%). Initial treatments included azoles, liposomal amphotericin B (AmBisome), and miltefosine (each 20%); cryotherapy and parenteral pentamidine (each 10%); with compounded topical paromomycin added in 15% of cases. 35% of individuals (n=7) failed initial treatment and required additional treatment courses due to persistent lesions (57%) or relapse following initial resolution (14%). 10% (n=2) required a third treatment course due to persistent/recurrent lesions. Conclusion While CL in travelers has a variety of clinical manifestations, travel history, knowledge of key clinical features, and lesion patterns can aid in early diagnosis and treatment. CL is associated with early treatment failure rate for uncertain reasons. Disclosures All Authors: No reported disclosures

ABSTRACT Recent decades have seen a rise in the incidence of tick-borne diseases in the US, along with an increased number of pathogens transmitted by ticks, and geographic expansion of tick populations. A variety of laboratory testing methodologies are available for the diagnosis of tick-borne diseases, including serology, microscopy, and molecular-based methods. The preferred approach varies by the specific disease, locally available test options, and the stage of illness at patient presentation. This mini-review focuses on updates in our understanding of the epidemiology of tick-borne diseases in the US and advances in the field of laboratory diagnostics.

Abstract The range of Babesia microti (Franca, 1910)-infected ticks is expanding, resulting in locally acquired human babesiosis cases occurring in new areas: Maryland (2009), the District of Columbia (2013), Virginia (2016), and West Virginia (2017). We collected host-seeking ticks from old fields, ecotones, forested habitats and animal hosts in Delaware, Maryland, and Virginia, 2010 to 2024. Ixodes scapularis Say, the tick vector of babesiosis, was captured in all 3 states. PCR revealed B. microti in 2.7% (36/1310) of I. scapularis, with site prevalence ranging from <1% to 12.5% infected. The first B. microti-positive I. scapularis was collected in Northampton County, Virginia, 2012. Of the B. microti-infected ticks, 50% (18/36) were coinfected with Borrelia burgdorferi and one was triple-infected with B. microti, B. burgdorferi, and Anaplasma phagocytophilum. We collected Ixodes keiransi Beati, Nava, Venzal, and Guglielmone ticks from Delaware and Virginia. We found B. microti and B. burgdorferi in those from Virginia, and B. burgdorferi in ticks from a shrew in Delaware. To our knowledge, this is the first report of B. microti and B. burgdorferi-positive I. keiransi from Virginia, and the first report of B. burgdorferi-positive I. keiransi from Delaware. Ixodes keiransi ticks rarely bite humans but are involved in the maintenance and spread of pathogens when sympatric with I. scapularis. We tested a subset of both tick species for Babesia duncani; none were positive. Jurisdictions in the southern mid-Atlantic region should expect babesiosis cases, and Lyme disease and anaplasmosis coinfections, and healthcare providers should consider these tick-borne infections as part of the differential diagnosis.

Abstract Background There is a lack of comprehensive, large cohort studies investigating the predictors for severity and hospitalization in human granulocytic anaplasmosis (HGA). Methods We conducted a retrospective cohort study including all cases of HGA identified by positive blood Anaplasma polymerase chain reaction (PCR) at the Mayo Clinic Laboratory, Rochester between 2011 and 2021. Multivariable logistic regression was performed to evaluate risk factors associated with hospitalization. Results A total of 465 cases were identified. Of those, 67% (n = 312) were managed in the outpatient setting. Hospitalized patients (n = 153, 33%) were more likely to be older (median age of 71 vs 61; P ≤ .001) and immunocompromised (17% vs 7%; P ≤ .001). The triad of leukopenia, thrombocytopenia, and transaminitis was observed in 24% of the cases, but was associated with risk of hospitalization (odds ratio [OR] 1.78, 95% confidence interval [CI]: 1.05–3.03; P ≤ .033). The presence of a coinfection did not impact mortality or hospitalization. Conclusions The risk factors for hospitalization in patients with HGA include altered mental status, higher absolute neutrophil count (ANC), advanced age, comorbidities, and immunosuppression. The classic hematological and biochemical profile may be absent in the majority of cases. Co-testing may be of higher benefit in select cases.

Context.— Generative artificial intelligence (GAI) is a promising new technology with the potential to transform communication and workflows in health care and pathology. Although new technologies offer advantages, they also come with risks that users, particularly early adopters, must recognize. Given the fast pace of GAI developments, pathologists may find it challenging to stay current with the terminology, technical underpinnings, and latest advancements. Building this knowledge base will enable pathologists to grasp the potential risks and impacts that GAI may have on the future practice of pathology. Objective.— To present key elements of GAI development, evaluation, and implementation in a way that is accessible to pathologists and relevant to laboratory applications. Data Sources.— Information was gathered from recent studies and reviews from PubMed and arXiv. Conclusions.— GAI offers many potential benefits for practicing pathologists. However, the use of GAI in clinical practice requires rigorous oversight and continuous refinement to fully realize its potential and mitigate inherent risks. The performance of GAI is highly dependent on the quality and diversity of the training and fine-tuning data, which can also propagate biases if not carefully managed. Ethical concerns, particularly regarding patient privacy and autonomy, must be addressed to ensure responsible use. By harnessing these emergent technologies, pathologists will be well placed to continue forward as leaders in diagnostic medicine.