bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemophagocytic Lymphohistiocytosis (HLH): A Clinical and Pathological Grading System Based on the Presence of Hemophagocytosis
Authors: Reid J , Demianets R , Rezk S
Published: October 2024

Introduction/Objective

Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous group of disorders resulting from uncontrolled immune activation leading to severe hyperinflammation. The syndrome can be primary (genetic) or secondary to infections, malignancies, or autoimmune diseases. The diagnosis of HLH is challenging due to its nonspecific clinical presentation and the overlap with other conditions. This study aims to establish a reliable diagnostic approach for HLH by evaluating the accuracy of bone marrow histomorphological criteria in diagnosing the disease.

Methods/Case Report

We conducted a retrospective analysis of clinical and pathological data from patients diagnosed with HLH over a 12year period and included 38 patients with suspected HLH. A grading system for hemophagocytosis was developed based on the extent and frequency of hemophagocytic activity observed in bone marrow biopsies. Hemophagocytosis is categorized into four grades: Grade 0 indicates its absence, Grade 1 describes mild hemophagocytosis with less than 2 histiocytes containing hemophagocytosis per slide, Grade 2 denotes moderate hemophagocytosis with 3 to 5 histiocytes per slide, and Grade 3 signifies severe hemophagocytosis with more than 5 histiocytes per slide. Clinical data, comprising laboratory parameters and treatment outcomes, were analyzed in relation to the histologic grading of hemophagocytosis.

Results (if a Case Study enter NA)

Among the cases, grade 1 had 1 resolved case, 4 cases meeting 2 criteria all resolved, and 13 cases meeting 4 or more criteria resulted in 5 deaths and 8 resolutions. Grade 2 included 10 cases, with 2 meeting 2 criteria and resolving, and 8 meeting 4 or more criteria resulting in 5 deaths and 3 resolutions. Grade 3 comprised 10 cases, with 3 meeting 3 criteria and resolving, 3 meeting 5 or 7 criteria resulting in 1 death and 2 resolutions each, and 4 meeting 6 or 8 criteria resulting in 2 deaths and 2 resolutions each.

Conclusion

In our retrospective analysis of bone marrow biopsies from 38 patients with suspected HLH, our results indicate that the presence and extent of hemophagocytosis strongly suggest HLH. Implementing these criteria significantly improved diagnostic accuracy, reducing misdiagnosis rates and enabling timely therapy initiation. This study underscores the value of bone marrow histomorphological criteria in diagnosing HLH and stresses their importance in the standard diagnostic process for suspected HLH cases.
NPM1 Minimal Residual Disease Detection at Any Time by Clinical Next Generation Sequencing Predicts Poor Survival in Acute Myeloid Leukemia
Authors: Sallman D , Xie Z , Chan O , Shao R , Song J , HUSSAINI M O
Published: October 2024

Introduction/Objective

Acute Myeloid Leukemia (AML) is a deadly disease that accounts for 80% of adult leukemia cases and carries a 5-year survival rate of only 31.7%. Historically, prognosis hinged heavily on cytogenetic stratification, but more recently minimal residual disease (MRD) has emerged as a crucial prognostic marker. Flow cytometry, PCR, and NGS can all be employed for MRD detection, but each carries its own limitations. Though technically challenging, NGS has the advantage being able to simultaneously assay multiple mutations, thus providing for broader applicability. NPM1 is the most prevalently mutated gene in AML (25-35%). In this study, one of the largest of its kind, we investigate the feasibility and impact of NPM1 MRD detection by clinical NGS.

Methods/Case Report

IRB approval was obtained. We reviewed the medical record and clinical laboratory data sets from AML patients treated at Moffitt Cancer Center who had NPM1 status assessed by clinical NGS, Testing was performed at LabPMM (San Diego, CA). DNA was extracted, and NPM1 exon 12 was amplified. PCR products were sequenced on an Illumina MiSeq. The clinically reportable assay sensitivity was 5 x 10-5 though lower levels were detected on review of raw data. Kaplan-Meier analysis was conducted for overall survival assessment. Since patients often had multiple tests, we divide patients into those with NPM1+ at any time and NPM1 always negative.

Results (if a Case Study enter NA)

There was a total of 140 patients. Overall, 346 tests were conducted, with an average of 2.5 tests/patient. There were 85 patients (60%) who underwent serial testing. MRD+ patients (n=80) had a mean age of 66 years, while MRD- patients (n=60) had a mean age of 58 years. MRD+ patients exhibited a mean overall survival of 29.3 months versus 43.6 months for MRD- patients (p=0.005.)

Conclusion

Through our study encompassing a large patient cohort of 140 patients and nearly 350 testing data points, we confirm the feasibility and clinical utility of NPM1 MRD evaluation by NGS. The significantly poorer survival in patients who were MRD+ at any time during their disease provides a scientific rationale for serial NGS testing to enable risk stratification of NPM1+ AML patients.
Unexpected Presentations of Histiocytic Disorders as Mass Lesions in the Central Nervous System and Spine
Authors: Cutshall H , Post G , Gokden M
Published: October 2024

Introduction/Objective

A number of neoplastic and non-neoplastic hematolymphoid lesions can involve the CNS and spine. The majority are lymphomas, but some are rare lesions that mimic primary neoplasms clinically, radiologically, and even pathologically. Here, we present our experience of such cases.

Methods/Case Report

Pathology archives were searched, and five cases representing different challenges were identified to review clinical, radiological, and pathologic features. Case 1. 31-year-old female presented with vision changes and hearing loss and numerous supratentorial extra-axial masses, suspicious for meningiomatosis.

Extensive lymphoplasmacytic infiltrate was seen at Intraoperative consult (IOC). Final diagnosis was Rosai-Dorfman disease with features of IgG4 related disease. Case 2. 31-year-old female with Chronic myeloid leukemia (CML), which progressed to AML presented with a right frontal brain mass. IOC displayed atypical hematolymphoid infiltrate. Final diagnosis was myeloid sarcoma. Case 3. 29-year-old male presented with a T10 vertebral lesion. IOC showed hypercellular tissue with atypia and was deferred. Subsequently, histiocytic sarcoma was diagnosed. Case 4. 22-year- old male presented with a T3-T4 intradural, extramedullary mass radiologically thought to be a meningioma. No IOC was requested. Final diagnosis was juvenile xanthogranuloma. Case 5. 36-year-old male presented with a C7-T1 dura-based mass. IOC revealed granulomatous inflammation, eventually resulting in a diagnosis of non-necrotizing granulomatous inflammation, suggesting sarcoidosis.

Results (if a Case Study enter NA)

NA

Conclusion

These cases highlight the importance of incorporating all clinical and radiological findings into pathologic evaluation, while being alert for mimickers and pitfalls. Both practicing general surgical pathologists and neuropathologists should remain alert to such unexpected and unusual cases especially at the time of IOC and initiate appropriate work-up with expedited diagnostic protocols such as intraoperative cultures, flow cytometry, and cytogenetics, as the differential diagnosis is broad with inflammatory, infectious, and neoplastic possibilities, presenting a variety of challenges.
“Synchronous Development of Acute Myeloid Leukemia and Multiple Myeloma: A Rare Case Presentation”
Authors: Alrammahi A , Ndam F , Snower D , Kapila N
Published: October 2024

Introduction/Objective

The co-occurrence of acute myeloid leukemia (AML) and multiple myeloma (MM) presents a diagnostic challenge, particularly when it arises without prior chemotherapy exposure. This study aims to elucidate the clinical characteristics, diagnostic approach, and management implications of such synchronous hematologic malignancies through a detailed case report.

Methods/Case Report

A 64-year-old female with no previous medical history presented with chest pain, shortness of breath, and hematologic abnormalities. Clinical evaluation revealed severe pancytopenia, cystic liver mass, enlarged retroperitoneal lymph nodes, and hypercalcemia. Peripheral blood smear indicated hematologic malignancy, prompting further investigation. Bone marrow aspirate analysis, including flow cytometry and immunohistochemical stains, was performed to confirm the dual cell populations of AML blasts and MM plasma cells.

Results (if a Case Study enter NA)

Flow cytometric analysis and immunohistochemical stains of the bone marrow aspirate confirmed the presence of distinct cell populations: blasts expressing markers consistent with AML and abundant plasma cells indicative of MM. Monotypism of plasma cells was established, with kappa predominance. Notably, there was no coexpression of markers between the AML blasts and MM plasma cells.

Conclusion

This case highlights the rare occurrence of synchronous AML and MM in a patient without prior chemotherapy exposure. The diagnostic approach involving flow cytometry and immunohistochemical stains proved essential in confirming the dual hematologic malignancies. Clinicians should be vigilant for such presentations, as timely diagnosis and appropriate management are crucial for optimizing patient outcomes in these complex cases.
Myeloid neoplasm with concurrent BCR::ABL1 translocation, and JAK2 and SF3B1 co-mutations with severe fibrosis: a case report
Authors: Hanan A , Safdar J , Khan A , Gur H , Lee J , Mahmood N , Karp L , Singh R , Shaham M , Spitzer S , Arif M , Cohen N
Published: October 2024

Introduction/Objective

Myeloproliferative neoplasms (MPNs) arise from hematopoietic stem cells with somatically altered tyrosine kinase signaling. Classification of MPNs is based on hematologic, histopathologic and molecular characteristics including the presence of BCR::ABL1 and JAK2 V617F.

Methods/Case Report

We present a 72-year-old male with a myeloid neoplasm and severe fibrosis, with concurrent BCR::ABL1 translocation, and co-mutations in JAK2 and SF3B1. Initially, the patient was diagnosed with anemia, thrombocytosis and folic acid deficiency and was prescribed supplemental folic acid. At follow up, normocytic anemia and thrombocytosis were seen. Bone marrow biopsy showed overtly fibrotic marrow with maturing trilineage hematopoiesis, markedly increased megakaryopoiesis and ring sideroblasts. Chromosome analysis and sequential GTG-fluorescence in situ hybridization (FISH) of metaphases using the dual-fusion three color probe BCR/ABL1- ASS1 revealed abnormal results. A t(9;22)(q34;q11.2) was detected along with an atypical FISH signal pattern with a single BCR::ABL1 fusion on the derivative chromosome 22 instead of the typical two fusions, resulting in loss of one BCR::ABL1 fusion signal including loss of the ASS1 on the derivative chromosome 9. Next-generation sequencing (NGS) analysis detected double driver mutations, JAK2 p. Val617Phe and SF3B1 p. Lys700Glu in the patient. JAK2 V617F mutation was further confirmed by real-time quantitative PCR (qPCR) with variant allele frequencies of 39.7%. Additionally, the patient was positive for BCR::ABL1 p190 fusion transcript (10%). The presence of JAK2 and SF3B1 co-mutations along with ring sideroblasts and the patient’s clinical presentation suggests the diagnosis of myelodysplastic/myeloproliferative neoplasm with thrombocytosis and SF3B1 mutation.

Results (if a Case Study enter NA)

NA

Conclusion

Although, the detection of t(9;22) by chromosome analysis is a diagnostic feature of chronic myeloid leukemia (CML), our case adds to the limited information on co-existence of BCR::ABL1 and JAK2 V617F with SF3B1 mutations in severe fibrosis. The frequency of co-occurrence, the temporal order of acquisition and clonal relationship of these alterations is poorly understood, and it is also unclear for this case since there is no prior bone marrow biopsy or diagnostic work up. Literature search of these rare cases show that some patients with similar findings progress to myelofibrosis as in this patient. It is possible that the presence of two mutated tyrosine kinase genes accelerates disease progression.
Ordering and Utilization of Peripheral Blood Flow Cytometry in an Academic Medical Center
Authors: Golab K E , Harrington A , Fydryszewski N , Schur C
Published: October 2024

Introduction/Objective

The American Society for Clinical Pathology and the American Board of Internal Medicine’s Choosing Wisely campaign have developed best practice utilization guidelines for laboratory testing. Wisconsin Diagnostic Laboratories and Medical College of Wisconsin leadership noticed an increase in inappropriate peripheral blood flow cytometry testing in the last two years. An intervention was developed to help reduce inappropriate testing requests.

Methods/Case Report

The laboratory implemented a clinical decision support tool in the electronic medical record system that looked at patient diagnosis, history of stem cell transplant, and recommendations from pathology to help improve utilization of peripheral blood flow cytometry testing. Based on patient history, flow cytometry testing would move forward, or an alternative order of a peripheral blood pathology smear review with reflex to flow cytometry was offered. Pathologists would reflex flow cytometry if abnormalities were present by light microscopy or predetermined reflexing criteria were met.

Results (if a Case Study enter NA)

314 cases were included for analysis. The intervention reduced volumes of peripheral blood flow cytometry in patients without a history of hematologic malignancy by 49.1% (p=<0.001). Appropriate utilization increased from 47.0% in the pre-intervention period to 80.4% in the post intervention period (p=<0.001). Significant diagnosis rate increased from 42.5% to 50.0% (p=.261). This resulted in a cost savings of $12,232 for the health system during the intervention period.

Conclusion

Clinical decision support based utilization efforts provide strong and consistent interventions to reduce the unnecessary and inappropriate ordering of peripheral blood flow cytometry.
Assessment of Molecular Response to Treatment of Chronic Myeloid Leukemia at Kigali University Teaching Hospital of Kigali
Authors: MUKANDEKEZI A
Published: October 2024

Introduction/Objective

Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative neoplasm that gives proliferation advantage to the granulocytes, it is one of the commonest chronic hematological malignancies and it is a burden to the world especially Rwanda. Since the introduction of Tyrosine Kinase Inhibitors (TKIs) especially the first line Imatinib in 2001, the outcomes of CML have been much improved and currently, in developed world, the life expectancy of patients with CML is almost the same as general population. However, it is not the case in developing world especially Rwanda due to limitation on access of all TKIs.

The aim of this study is to assess molecular response on Imatinib of patients with CML.

Methods/Case Report

This was a one arm open label prospective cohort study that recruited 31 patients with CML and on Imatinib for at least three months and followed for 11 months. The clinical features at diagnosis and recruitment were captured from the hospitals filing systems and investigator’s own assessment at the time of recruitment respectively. Information was filled to a well-structured questionnaire. Lab results at the time of diagnosis was obtained from laboratories reporting systems. Samples for full blood count and BCR-ABL1/ABL1 were withdrawn from the patients at the time of diagnosis and they were run at University Teaching Hospital of Kigali (CHUK). Data analysis was performed using Statistical Package for the Social Sciences (SPSS 28) and South Texas Art Therapy Association (STATA 15.0).

Results (if a Case Study enter NA)

Better response according to European LeukemiaNet (ELN) defined as optimal response was found among only 38.7% and resistance to imatinib defined by ELN as Failure and warning was found in 45.2% and 16.1% respectively. The eleven months survival rate was 93.5% in which it is 85.7% among those in treatment failure group. Late presentation showed by presence of very high White Blood Cells above 100x109/L and massive splenomegaly at diagnosis, and poor adherence to Imatinib according to Morsiky Medication Adherence Scale (MMAS-8) are associated with poor molecular response to Imatinib.

Conclusion

CML is a serious disease especially to those who suffer from it; on time diagnosis, access to all available lines of TKIs and better follow up are crucial for patients with CML to access the same life expectancy as general population as it is in developed world.
Can multiplexing Immunohistochemistry for PD-L1, CD68, and CD3 improve scoring for “Keytruda®” Indication?
Authors: Melvin S , Nam M
Published: October 2024

Introduction/Objective

FDA-approved companion diagnostics immunohistochemistry scoring for PD-L1 is essential for Keytruda ® treatment in patients with NSCLC. The challenge with PD-L1 testing alone is false positive PD-L1 expression score due to alveolar macrophage infiltration. Multiplexing with PD-L1, CD68, and CD3 could help alleviate this issue. By viewing PD-L1 expression on tumor cells, CD68 expression on macrophages, and the presence of CD3+ T cells on a single slide, pathologists will be able to see the presence of an immune microenvironment (high PD-L1 expression with abundant macrophages) and the potential for response to other types of Immunotherapies (presence of T cells).

Methods/Case Report

Positive NSCLC FFPE blocks were sectioned and stained using Agilent Dako Omnis. Two combinations of multiplex staining were prepared for each block by staining PD-L1 (brown) alone first followed by CD3 and CD68. First slide: CD3(Vina Green), CD68(Magenta); Second slide: CD3(Magenta), CD68(Vina Green). To prevent wash out, dehydration and clearing occurred with two changes of 100% alcohol and three changes of xylene for manual cover slipping.

Results (if a Case Study enter NA)

Manual scoring of PD-L1 alone vs two different multiplex combinations was compared to Agilent Proscia digital AI scoring using a preliminary study set (9 positives, 2 negatives). CD68-green and CD3-magenta showed better discrimination than CD68-magenta and CD3-green at a glance. This could be due to the staining intensity and nonspecific background staining of CD68-magenta. Manual scoring was higher compared to digital scoring, which could be due to the human error of positive bias. Digital scoring was significantly lower for PD-L1 alone vs both multiplex stains. The digital scoring for the multiplex stains showed a higher scoring percentage by removing false positive macrophages.

Conclusion

Reflex scoring workflow is being developed to focus on difficult cases using the multiplex triple stain, i.e. clear negative and clear >50% with 3+ intensity. More cases will be collected and studied to further investigate the improved PD-L1 scoring with digital imaging assisted by artificial intelligence and to compare the multiplex color combinations.
P53 ABNORMAL ENDOMETRIAL CANCERS – ANALYSIS OF PD-1 CO-EXPRESSION BY CD4 HELPER AND CD8 CYTOTOXIC T-CELL SUBTYPES IN THE TUMOR MICROENVIRONMENT
Authors: Ariyanayagam S , Baksh D
Published: October 2024

Introduction/Objective

Endometrial cancer is classified into four molecular subtypes of which the p53 abnormal variant is biologically aggressive. Programmed cell death protein 1 (PD-1) is a cell surface receptor and a vital inhibitor of the immune response. Programmed death ligand 1 (PD-L1) is a trans-membrane co-inhibitory factor of the immune response. PD-1 / PDL-1 interaction inhibits the activation and survival of T cells and decreases cytokine secretion.

Some cancer cells express PD-L1, which then bind to PD-1 expressed on tumor specific T cells, and escape destruction by the T cells. The CD4/CD8 T cell immune response in the p53 abnormal variant has not been adequately evaluated with respect to the PD-1/PD-L1 pathway. Understanding this immune microenvironment is important to assess the potential use of anti-PD-1 targeted tumor therapy.

Methods/Case Report

Twenty cases of p53 abnormal endometrial cancers were obtained. All cases were PDL-1 +ve by immunohistochemistry. Two dual stains were performed - PD-1/CD4 and PD-1/CD8 antibodies.

Six high-power fields (40x objective) with the greatest concentration of lymphocytes were counted. The total number of CD4 or CD8-only cells was recorded for each of the two stained slides for each case. The total number of PD- 1/CD4 and PD-1/CD8 positive cells were counted and recorded.

The results were tabulated, and a paired t-test statistical analysis was performed to compare the total number of CD4 and CD8 cells and the percentage of PD-1/CD4 cells with the percentage of PD-1/CD8 positive cells.

Results (if a Case Study enter NA)

A statistically significant increase in the number of CD8 (Mean = 383) compared to CD4 cells (Mean = 317) was seen in the tumor microenvironment (p-value=0.0023). Evaluating PD-1 expression, the converse was true, and the percentage of PD-1/CD4 cells (Mean = 13.035%) was increased compared to the percentage of PD-1/CD8 cells (Mean = 9.205%), achieving statistical significance (p-value=0.0116).

Conclusion

The statistically significant increase in CD8 cells in the tumor and its stroma suggests that a robust cytotoxic response is being mounted to this high-risk molecular variant. The concurrent increase in CD4/PD-1 positive lymphocytes provides an appropriate microenvironment for the use of anti-PD-1 immunotherapy, since blockade of PD-1 on CD4 helper T cells may augment the tumor ablative cytotoxic properties of the CD8 positive lymphocytes.

This includes reinvigoration of exhausted CD8+ T cells. Anti-PD-1 may also prevent the inhibition of PD-1 positive CD8 cells.
Relocating a highly complex 5 million test per year hospital based laboratory - what you should know and what you should avoid
Authors: ZURETTI A
Published: October 2024

Introduction/Objective

A highly complex hospital based laboratory performing 5 million test per year in New York City was commanded to relocate due to a clinical department need to expand. This presented a challenge regarding the obtention of a suitable new space within the existing facility, the architectural design to optimize the performance of both Anatomic and Clinical Pathology, the choice between acquirring brand new instrumentation versus relocation of the existing instruments. At the same time, we had to interact with hospital wide Facilities Management and Information Services/ Electronic Medical records. In addtion, for quality improvement and to alleviate the shortage of licensed technologist in our state, it was decided that a robotic line was to be implemented in the new facility.

Methods/Case Report

Several space options were assessed. Once the most suitable space was found a decision was made to move in two phases, Anatomic Pathology first, and subsequently Clinical Pathology. New work stations were purchased for Anatomic Pathology. New instruments were obtained for Chemistry, Immunology and Hematology. All instruments were validated.. Our laboratory information system had to be upgraded in order to support the robotic line, which is to be implemented after the Clinical Laboratory move. This will facilitate the expansion of in house provision of laboratory services to outpatient facilities / clinics expandin our outreach. The new space includes room to expand as well as abundant swing space. Our next phase includes the relocation of our Blood Bank and expansion of our Microbiology Services

Results (if a Case Study enter NA)

Due to delays in the obtention of the State Occupational permit the move was postponed several times.

It was decided to move in two phases, once the permit was received.

The Laboratory successfully moved in a two week period, and was fully operational overnight.

Conclusion

The Laboratory successfully moved in a two week period, and one week later we underwent a routine State inspection, in which the rigourous inspectos encountered very few deficiencies. The Laboratory is fully operational to the satisfaction of our clinical departmenst and preparing to the next phase including Blood bank and expanding our outreach program
Enhancing Laboratory Outcomes Through “Stacked” Professional Credentials: A Study of ASCP BOC Trends
Authors: Baker J B , Spiczka A
Published: October 2024

Introduction/Objective

Laboratory medicine and pathology are rapidly evolving, requiring laboratory professionals to continuously adapt and enhance their skills to ensure high standards of patient care. The American Society for Clinical Pathology (ASCP), Board of Certification (BOC) has observed a notable increase in the number of credential holders obtaining multiple, or stacked, BOC credentials. This phenomenon is not limited to the U.S. but is also evident in various countries globally. This study aims to explore the trends of stacking credentials among BOC credential holders, examining its implications for professional development, career progression, and the overall impact on laboratory outcomes.

Methods/Case Report

This study utilized a comprehensive analysis of ASCP BOC’s credentialing data, focusing on individuals with multiple credentials. Data were segmented by degree (Bachelor’s, MBA, PhD, etc.), credential count (ranging from 2 to 10), and geographical distribution across the U.S. and internationally. The analysis sought to identify patterns, growth trends, and the professional demographics most inclined towards obtaining stacked credentials.

Results (if a Case Study enter NA)

The study revealed that approximately 11% of BOC credential holders possess two or more credentials, indicating a significant trend towards continuous professional development and specialization. The breakdown of stacked credentials by degree shows a higher percentage propensity among individuals with MBAs (as one degree type). Geographical analyses highlighted hotspots for professionals seeking multiple credentials both at the domestic U.S. level and internationally.

Conclusion

This suggests the BOC, and by extension the laboratory medicine and pathology community, can greatly benefit from promoting and facilitating the acquisition of multiple credentials. By doing so, the field can ensure a workforce that is not only highly qualified but also adaptable to the changing dynamics of healthcare, thereby reinforcing the indispensable role of laboratory professionals in the broader healthcare ecosystem.
Optimizing RT-qPCR Detection of Enveloped and Non-enveloped Viruses in Wastewater: A Comparative Study in Grenada
Authors: Farmer-Diaz K , Matthew-Bernard M , Macpherson C , Ramos-Nino M , Cheetham S , Mitchell K
Published: October 2024

Introduction/Objective

Wastewater-based epidemiology (WBE) is an effective methodology to monitor the community spread of pathogens and has successfully been used to identify potential outbreaks. Most standardized methodologies used in WBE for the detection of viruses have been validated for non-enveloped viruses; therefore, there is a need for the development of a standardized protocol for the detection of both enveloped (such as SARS- CoV-2) and non-enveloped viruses (such as Hepatitis A). Wastewater seeded with Pseudomonas syringae bacteriophage (Φ6), an enveloped virus surrogate, and coliphage MS2, a non-enveloped virus surrogate, were used to evaluate the efficiency of four different aluminium flocculation methods. Untreated wastewater spiked with known amounts of surrogates was used as a control for viral recovery.

Methods/Case Report

Four different viral concentration methods were used.

Method 1: AlCl3 flocculation at pH 3.5 followed by centrifugation, resuspension with 10% beef extract and filtered with a 0.22 μm filter.

Method 2: AlCl3 flocculation at pH 6.0 followed by room temperature agitation, centrifugation, and resuspension with 3% beef extract.

Method 3: Mixed method protocol-AlCl3 flocculation at pH 3.5 followed by room temperature agitation, centrifugation, resuspension with 3% beef extract.

Method 4: Mixed method protocol-AlCl3 flocculation at pH 6.0 followed by centrifugation, resuspension with 10% beef extract filtered with a 0.22 μm filter.

Viral RNA was extracted after concentration, and reverse-transcription quantitative polymerase chain reaction (RT- qPCR) was performed.

Results (if a Case Study enter NA)

The average Ct reduction of untreated wastewater compared to spiked was calculated for each method. The highest recovery of both Φ6 and MS2 was obtained using Method 3 (mixed protocol) with a significant difference at 95% confidence in the recovery of Φ6 (p= 0.0029) and MS2 (p= 0.0225).

Conclusion

Previous results from our lab demonstrate the importance of pH during the flocculation step of viral concentration using different methods of concentration. Here, the results suggest that the increased contact time between virus and the flocs is also critical. This can facilitate the implementation of WBE in Grenada to monitor and track potential viral outbreaks of both enveloped and non-enveloped viruses such as SARS-CoV-2 and Hepatitis A.
Molecular epidemiology of diabetic foot ulcers
Authors: Mensah S
Published: October 2024

Introduction/Objective

The study accessed the prevalence of Escherichia coli in diabetic diabetic foot infection(DFI) and diabetic foot osteomyelitis(DFOM).A single retrospective single centre was conducted on patients diagnosed with E.coli isolated from deep DFI and DFOM at Komfo Anokye Teaching Hospital over 2 year period.

Methods/Case Report

Phylogenetic backgrounds,virulence factors(VFs) and antibiotic resistance profiles were determined.Whole genome analysis of E.coli strains isolated from same periods were performed.

From two years study period,35 E.coli strains isolated from 33 patients were analysed,73% were isolated from DFOM

Results (if a Case Study enter NA)

The majority of the strains belonged to the virulent B2 and D phylogenetic groups(82%).These isolates exhibited a significant higher average of VFs number greater than strains belonging to the other groups (p<0.0001).papG2 gene was significantly more detected in strains belonging to B2 phylogeny isolated from DFI compared to DFOM(p=0.003)

The most prevalent antibiotic resistance pattern was observed for ampicillin(82%)cotrimoxazole(45%) and ciprofloxacin (33%).The genome analysis of strains isolated at two periods in DFOM showed a decrease of the genome size and this decrease was more important for the strains isolated at 9 months(versus 4 months)

Conclusion

Shared mutation on the putative acy-CoA dehydrogenase-encoding gene aidB was observed on both strains. E.coli isolated from DFOM werA shared highly genetically diverse with different pathogenicity traits.Their adaptation on the bone structure could require genome reduction and some important modifications in the balance virulence and resistance of the bacteria
Klebsiella pneumoniae Isolated from Critically Ill Patient with Nosocomial Pneumonia Elicits a Proteinopathy
Authors: Hanna S , Pankratz K , Pham A , Dickey Z , Morrow A , Voth S , Morrow R , Wagener B , Pittet J , Choi C , Lin M , Stevens T
Published: October 2024

Introduction/Objective

Gram-negative opportunist Klebsiella pneumoniae has been identified by the World Health Organization as a critical threat partially due to its prevalence in nosocomial pneumonia. Survivors often suffer increased long-term morbidity and mortality secondary to end-organ damage, including impaired cognition.

Pseudomonas aeruginosa lung infection generates an endothelial-derived tauopathy, yet whether K. pneumoniae infection elicits this phenomenon is unknown. We hypothesize K. pneumoniae infection triggers pathogenic tau release from endothelial cells, contributing to an infection-induced proteinopathy.

Methods/Case Report

We utilized wildtype, control, and tau-/- pulmonary microvascular endothelial cells (PMVECs). Tau was monitored via immunoblotting. K. pneumoniae (Kp 1-008) was isolated from the bronchoalveolar lavage of a critically ill patient diagnosed with a monomicrobial nosocomial pneumonia. Supernatants were filter-sterilized and boiled prior to transfer to naïve cells to determine transmissibility. Barrier permeability was assessed via transwell assays, cell viability through resazurin, and amyloid burden was monitored with Congo Red and Thioflavin T.

Results (if a Case Study enter NA)

Kp 1-008 infection induced permeability and tau release from control cells in a dose-dependent manner. Tau and amyloid burden increased over time in supernatants from control PMVECs. Sterile infection supernatants from control cells increased permeability and impaired oxidative metabolism in naïve cells whereas supernatants from tau-/- cells were markedly less pathogenic. Kp 1-008 induced proteinopathic cytotoxicity persisted strongly through two passages when derived from control cells but one passage when produced from tau-/- PMVECs.

Conclusion

Our results suggest that 1) K. pneumoniae infection of PMVECs generates a lung endothelial-derived proteinopathy and 2) endothelial tau is a significant contributor to its virulence.
A Retrospective Analysis of Broad-Range Microbial PCR-Sequencing Findings and Culture Results in 101 Bronchoalveolar Lavage Specimens
Authors: Hamid A , Qin X
Published: October 2024

Introduction/Objective

Pneumonia is a significant cause of illness and death, identifying its cause is challenging and critical for effective antibiotic treatment and for reducing the length of stay. Bronchoalveolar lavage is commonly performed to obtain high-quality lower respiratory tract samples. While traditional culture methods have been the standard for pathogen detection, microbial rRNA broad-range polymerase chain reaction (BR-PCR) has emerged as an alternative method.

Methods/Case Report

A retrospective analysis was conducted on 101 patients suspected of severe pulmonary infections at Oregon Health and Science University (OHSU).

Results (if a Case Study enter NA)

When important microbial etiology is considered, culture detected 26%, while BR- PCR detected 20%. Both methods proved effective in detecting common pathogens; however, culture demonstrated superiority in detecting fungal pathogens due to potential sampling biases associated with PCR. Furthermore, culture holds the advantages of being faster and more cost-effective, although it may fail to detect difficult to non-cultivable organisms, like Legionella spp. Culture is indispensable for susceptibility testing and strain typing purposes. More often than not, BR-PCR captures airway microbiota indiscriminately with minimal clinical significance, potentially confusing clinical interpretation.

Conclusion

Novel technologies like Biofire Pneumonia Panel (BPP), a multiplex PCR, have shown promise in the rapid detection of pneumonia pathogens including bacteria, viruses, and resistance genes (< 2 hours), complementing the traditional culture methods for microbiological diagnosis. Based on this analysis we recommend introducing BPP at OHSU.
Targeted microscopic rare cell retrieval yields sufficient deoxyribonucleic acid for molecular testing in fixed tissue with low tumor fraction
Authors: Fakhari M , Mullane P , Gu W , Dong F , Lowe A C
Published: October 2024

Introduction/Objective

DNA extracted from formalin-fixed paraffin-embedded tissue (FFPE) blocks is often utilized for molecular oncologic testing. Cases with low tumor fraction often fail to obtain adequate tumor for molecular testing.

We hypothesized that targeted retrieval of visualized tumor cells from FFPE sections, including those with low tumor fraction, could yield sufficient DNA for molecular testing.

Methods/Case Report

Unstained sections of various thicknesses were cut from low tumor fraction blocks and visualized by light microscopy and the RareCyte CyteFinder II (Seattle, WA). Microscopic cell retrieval was performed on the RareCyte using 40 µm diameter needles from identified regions of interest. Samples were deparaffinized using Zymo Research (Tustin, CA) solution, and digested and purified using Zymo-Spin Column technology. Sample DNA was quantified with NanoDrop Spectrophotometer (Chino, CA).

Results (if a Case Study enter NA)

Regions of interest were morphologically identifiable on 4-10 µm thick sections and harder to visualize on 12 µm thick sections. Targeted microscopic cell retrieval was performed with 40 µm diameter needles. Retrieval was performed from 26 to 34 regions of interest per sample and pooled for molecular testing. Samples (n=4) yielded a mean of 81.0 ng of DNA ± 18.4 ng (1.8 ng/µL ± 0.4 ng/µL) and a median of 87.75 ng (1.95 ng/µL).

Conclusion

Targeted microscopic cell retrieval from low tumor fraction FFPE slides using the RareCyte CyteFinder II can provide sufficient DNA material for molecular testing. Application of this technology has the possibility of recovering sufficient tumor DNA for molecular testing from specimens that may be deemed unsuitable for molecular testing according to current standards. We aim to focus on increasing sample size and completing comprehensive molecular analysis as we continue to expand this work.
Transforming growth factor-beta and Epithelial-mesenchymal transition is considered an attractive pivotal Genetic-Biomarker for Hepatocellular carcinoma with hepatitis C virus-infection patients
Authors: Fyala A S , Sultan A
Published: October 2024

Introduction/Objective

Hepatocellular carcinoma (HCC) is the most common cause of cancer worldwide. HCC is induced by hepatitis C-virus (HCV) which represents a major health problem in Egypt. The transforming growth factor- beta (TGF-β) is a potent cytokine that has a multifunctional pro-fibrotic activity, for enhancing liver-inflammation, fibrosis, tumor-progression, and metastasis. TGF-β has a vital role in the regulation of tumorigenesis, by controlling epithelial-mesenchymal-transition (EMT), apoptosis, proliferation, and differentiation process. The dysregulation of TGF-β, Wnt/β-catenin signaling pathways, by HCV-core protein is implicated in the development of HCC.

Methods/Case Report

The study aims to determine the effect of the HCV-infection in HCC patients with a set of growth-factors, EMT, and anti-apoptotic protein expression levels by using western blotting and immunohistochemistry staining analysis.

Results (if a Case Study enter NA)

These results observed an insignificant increase in the expression of TGF-β protein for HCC/HCV-infected cells compared to the HCV-uninfected cells, and the expression of the β-catenin protein level is elevated in the nucleus. Also, it showed that Bcl-2 protein is a significant increase in HCV-infected when compared to HCV-free groups. Positive staining for p53 protein was detected in the HCC/HCV-infected, more than in HCC/HCV-uninfected cells.

This data indicated that the core-protein of HCV can switch TGF-β from a tumor suppressor to a tumor promoter by suppressing apoptosis of hepatocytes and increasing EMT expression by activation of Wnt/β-catenin signaling pathway. Therefore, HCV-core protein enhances the nuclear accumulation of β-catenin expression protein to activate downstream target genes, which regulate cell-growth and cell-cycle progression.

Conclusion

It is speculated that HCV may exert an anti-apoptotic effect, and thus enhance tumorigenesis. Also, the accumulation of nuclear Wnt/β-catenin and p53 correlates with TGF-β expression, typically a late event in hepato- carcinogenesis. Taken together, the expression level of TGF-β, Wnt/β-catenin, Bcl-2, and p53 genotype in HCV is considered an indicator of shortened survival in patients, and may be an attractive clinically pivotal genetic biomarker to mediate liver pathogenesis.
Beamion LUNG-1, a Phase Ia/Ib trial of the HER2-specific tyrosine kinase inhibitor, zongertinib (BI 1810631), in patients with HER2-driven tumors: outcomes according to HER2 aberration type
Authors: Berz D , Heymach J , Opdam F , Barve M , Tu H , Wu Y , Mueller A M , Schroeter L , Sadrolhefazi B , Yoh K , Yamamoto N
Published: October 2024

Introduction/Objective

Beamion LUNG-1 (NCT04886804) is an ongoing, Phase Ia/Ib trial evaluating the novel HER2- specific tyrosine kinase inhibitor, zongertinib, in patients with HER2 aberration-positive solid tumors (Phase Ia) and HER2 mutation-positive NSCLC (Phase Ib). Here, we report data from Phase Ia according to HER2 aberration type.

Methods/Case Report

Phase Ia enrolled patients with HER2 aberration-positive (by local assessment: gene mutations, rearrangements, amplification, or overexpression) advanced/metastatic solid tumors. Patients received escalating doses of zongertinib BID (≥15 mg) or QD (≥60 mg), guided by a Bayesian model with overdose control.

The primary endpoint of Phase Ia was maximum tolerated dose (MTD) based on dose-limiting toxicities (DLTs).

Results (if a Case Study enter NA)

At data cut-off (January 29, 2024), 83 patients received zongertinib (BID n=17; QD n=66). Three patients had DLTs during the MTD evaluation period (G3 decreased platelets, G3 diarrhea, G4 neutropenia); the MTD was not reached. Treatment-related adverse events (TRAEs; all/G3/G4/G5) were reported in 76%/8%/1%/0% of patients. Confirmed investigator-assessed ORR/DCR rates in evaluable patients/patients with NSCLC (n=74/n=41) were 35%/85% and 44%/93%; median duration of response was 12.7/15.8 months, respectively. Based on preliminary data, 43 patients were HER2 mutation-positive (TKD/non-TKD/unknown n=36/n=6/n=1; A775_G776insYVMA n=16; G778dupGSP insertions n=3; S310F n=2); 23 patients overexpressed HER2 (2+/3+); 17 had HER2 amplification; 3 patients had NRG1 fusions. ORR/DCR rates (regardless of confirmation) in patients with TKD mutation-positive, A775_G776insYVMA-positive, non-TKD mutation-positive, G778dupGSP-positive and S310F- positive tumors were 64%/97%, 69%/94%, 20%/100%, 33%/100% and 50%/100%, respectively. ORR/DCR rates (regardless of confirmation) in patients with HER2 overexpressing tumors (+/- amplification) were 39%/83%. Of the 3 patients with NRG1 fusions, one had a partial response and two had stable disease.

Conclusion

In this preliminary analysis, zongertinib showed promising activity across a broad range of tumor types with different HER2 aberrations, including aberrations that have historically been difficult to treat. Updated data will be presented.
Comprehensive Approach to TB Diagnosis Powered by Machine Learning in Endemic Countries
Authors: Khan T , Khan I H , Ravindran R
Published: October 2024

Introduction/Objective

Tuberculosis (TB), a leading cause of mortality worldwide, is primarily diagnosed through methods that face numerous challenges, including lengthy result times and diagnostic inaccuracy, particularly in the context of drug-resistant strains and latent infections. This study proposes a novel approach that integrates multiple diagnostic modalities with machine learning to enhance TB diagnosis accuracy.

Methods/Case Report

The Machine learning model was tailored for the classification of radiological images, specifically CT scans, using original DICOM images to preserve valuable voxel information. The study involved a curated dataset of CT scans from Pakistan, TB-positive (smear+/Culture+ n=72; smear+/culture- n= 3; smear-/Culture+ n=16; smear-/Culture- n=14; clinical and radiological diagnosis only n= 12) and healthy (n=103) scans from COVID-CTSet were included in the training to assess the specificity of the model. Data preprocessing included noise reduction, histogram matching, and data augmentation techniques to ensure a robust training dataset. The methodology also incorporated using k-fold cross validation and a custom learning rate scheduling strategy to optimize the model’s training process. To further test its robustness, the model was evaluated by swapping random patches and analyzing 60 mm patches from the entire CT scan for local feature categorization.

Results (if a Case Study enter NA)

The model has demonstrated outstanding performance, with sensitivity and specificity exceeding 95% on our internal dataset (n=117) and aligning well with clinical, smear, culture, and anti-TB antibody findings. It also showed promising results on the external NIH TB Portal’s dataset (n= 1,460). The model is robust to variations in CT scanner image quality and noise profiles, maintaining strong generalization across datasets. Even when analyzing random 60mm patches, effectively classifying all local features

Conclusion

Our study enhances tuberculosis diagnostic methods in endemic regions by blending machine learning with conventional diagnostic techniques. This approach improves TB detection, particularly the early and accurate identification in areas impacted by drug-resistant strains and asymptomatic latent infections. By integrating radiological and clinico-pathological data, we aim to develop a comprehensive diagnostic score. Moving forward, we plan to further integrate this method into clinical decision support systems and adapt the approach to other infectious diseases.
Efficiently Improving Laboratory Workflow with Continuous Sample Loading by Validating the COBAS 5800 for HIV and HCV Viral Loads
Authors: Petersen J M , Jhala D
Published: October 2024

Introduction/Objective

A regional veteran affairs medical center (VAMC) was using the COBAS AmpliPrep/COBAS Taqman (Roche, Basel Switzerland) with a throughput of 72 tests/8 hours without continuous loading capability.

However, a newer cobas 5800 System (Roche, Basel Switzerland) was validated to allow for continuous loading with better parameters for workflow including 144 tests/per 8 hours with a capacity for continuous loading of samples without batching. As validation experiences between the two instruments is sparse in the literature, the validation is described.

Methods/Case Report

The validation process included a linearity study using 24 samples with levels (100, 600, 6,000, 60,000, 600,000, 2,000,000, and 6,000,000 copies/mL for HIV and 100, 300, 3,000, 30,000, 300,000, 3,000,000, and 30,000,000 copies/mL for HCV) across the analytical range, a precision study at a low (1,000 copies/mL) and high (100,000 copies/mL for HIV, 1,000,000 copies/ML for HCV) values of a triplicate run over 5 days, an accuracy study based on the precision study, a limit of detection study using 20 control samples at the limit of detection, control monitoring (high and low positive controls) over 9 runs, and a method correlation between the Cobas 5800 and the Cobas AmpliPrep/COBAS Taqman.

Results (if a Case Study enter NA)

The validation study resulted as expected. For the method correlation, the results were plotted and evaluated with an R2 of 0.937 (HIV from 91 samples) and 0.9646 (HCV from 74 samples). Linearity across the analytical range had an R2 of 0.9967 (HIV) and 0.9950 (HCV). All samples were detected at the limit of detection study. Both the accuracy and precision yielded expected results. The values from running the controls (high and low positive) over 9 runs remained within the expected range and within two standard deviations for all runs.

Conclusion

The COBAS 5800, which has improved capabilities compared to the COBAS AmpliPrep/COBAS Taqman, was validated for clinical use successfully.