Біологічний матеріал, необхідний для приготування мікропрепаратів, у діагностиці інфекційних і паразитарних захворювань людини

A6-year-old male castrated Greyhound was presented to the William R. Pritchard Veterinary Medical Teaching Hospital, University of California, Davis, with a 4-week history of apparent spinal pain and paraparesis. There was no improvement despite treatment consisting of oral carprofen (unknown dose) for 10 days followed by a tapering course of oral prednisone for 6 days before referral (1.2–0.2 mg/kg/d). Abnormalities on physical examination included 5% dehydration, a large flaccid bladder that was easily expressed, moderate conjunctivitis, and mild generalized patchy alopecia. On neurological examination, the dog was ambulatory with marked paraparesis and absent conscious proprioception in both pelvic limbs. The patellar, gastrocnemius, and pelvic limb withdrawal reflexes were decreased bilaterally, the cutaneous trunci reflex was absent caudal to L4, and the perineal reflex and anal tone were absent. The tail was flaccid with intact superficial pain sensation. The dog was painful with dorsal palpation of the lumbar spine. The neurological abnormalities were consistent with an L4–Cd myelopathy. A CBC and serum biochemistry had mild abnormalities, interpreted as clinically insignificant. Urinalysis indicated a specific gravity of 1.048 and marked proteinuria (4+ on sulfosalicylic acid method). Thoracic radiographs and abdominal ultrasound examination indicated no clinically relevant findings. After vertebral column radiographs, where no abnormalities were seen, two 22 G 3.5-in. spinal needles were placed, 1 at the L4–5 and 1 at the L5–6 intervertebral disc spaces, but cerebrospinal fluid (CSF) could not be obtained. CSF was collected from the cerebellomedullary cistern. A diagnostic myelogram was not performed and, therefore, magnetic resonance (MR) images of the lumbar spinal cord followed by those of the brain were acquired with a 1.5-T scanner.a T1-weighted (T1W) images and T2-weighted (T2W) images included the L3 vertebra to the caudal vertebrae and the entire brain to the C3 vertebra. The spinal cord was enlarged and slightly elevated and dorsoventrally flattened from the L4 through the S2 vertebrae. On the T2W images, there was intramedullary hyperintensity of the lumbar and sacral segments of the spinal cord and the cauda equina, with partial loss of epidural fat signal and gray and white matter definition in the mid lumber region (Fig 1A). After IV contrast administration,b there was moderate parenchymal contrast enhancement and mild diffuse meningeal enhancement from the cranial lumbar region to the conus medullaris (Fig 1B and C). On the postcontrast T1W images of the brain, there was diffuse meningeal enhancement, most evident along the tentorium cerebelli and the falx cerebri (not shown). (A) T2-weighted sagittal magnetic resonance images (MRI) from dog 1. Note the intramedullary hyperintensity compared with spinal cord gray matter, loss of epidural fat signal, and gray/white matter definition. (B) T1-weighted sagittal MRI and (C) T1-weighted sagittal MRI postcontrast from dog 1. There is moderate parenchymal contrast enhancement and mild diffuse meningeal enhancement (arrow) from L4 to the conus medullaris. The CSF had a markedly increased nucleated cell count (660 cells/μL; reference range, ≤ 3 cells/μL) and a moderate increase in the protein concentration (72 mg/dL; reference range, ≤ 25 mg/dL). The differential cell count was 15% neutrophils, 12% lymphocytes, 68% large mononuclear cells, and 5% eosinophils. On cytologic evaluation, most of the large mononuclear cells were considered typical macrophages; however, some of the cells were extremely large and variably vacuolated with large immature nuclei (Fig 2). Within this small subpopulation of large immature cells, numerous mitoses were noted (2, 3). There were occasional large multinucleate tumor cells, with a marked anisokaryosis of the component nuclei and nuclear fragmentation (Fig 3). Rare bizarre multinucleate tumor cell variants also were present (Fig 4). The interpretation was marked, mixed, predominantly histiocytic inflammation with underling neoplasia, most likely histiocytic sarcoma. Cerebrospinal fluid cytospin slide, dog 1. There is a marked, predominantly large mononuclear pleocytosis. Note the scattered, extremely large, single round cells with large immature nuclei. Within this small subpopulation of large immature cells, there are numerous mitoses. Wright–Giemsa stain, scale bar = 50 μm. Cerebrospinal fluid cytospin slide, dog 1. Occasional large multinucleate tumor cells are present that have marked anisokaryosis of component nuclei and nuclear fragmentation. Wright–Giemsa stain, scale bar = 30 μm. Cerebrospinal fluid cytospin slide, dog 1. Rare bizarre multinucleate tumor cell variants are present that also have marked anisokaryosis of component nuclei and nuclear fragmentation. Wright–Giemsa stain, scale bar = 30 μm. Treatment with an anti-inflammatory dosage of dexamethasone sodium phosphate IV (0.22 mg/kg/d) was initiated immediately, but the dog deteriorated neurologically over the next 36 hours after anesthesia and became nonambulatory with marked apparent spinal pain. Immunophenotyping of the cells in the CSF was carried out by staining CSF cytospin slides using canine-specific monoclonal antibodies against CD1c, CD11b, CD11c, CD11d, CD3, CD79a, CD34, pancytokeratin, vimentin, and a negative control using previously described methods.1 The large immature cells strongly expressed CD1c (Fig 5) and CD11c. The neutrophils expressed CD11c. As expected, all leukocytes expressed vimentin. The diagnosis was histiocytic sarcoma with secondary marked, mixed inflammation. Based on the deteriorating neurologic status and the poor prognosis associated with histiocytic sarcoma, the dog was euthanized and submitted for necropsy. Cerebrospinal fluid cytospin slide, dog 1. The large immature single round tumor cells have a variable, predominantly cytoplasmic membrane, expression of CD1c. Immunocytochemistry stain, AEC substrate, hematoxylin counterstain, scale bar = 30 μm. At necropsy, clinically relevant findings on gross and histological examination were restricted to the central nervous system (CNS). The meninges of the brain and the spinal cord were diffusely opaque. On the cut surface of the spinal cord, the subarachnoid space was diffusely filled by an infiltrative, pale tan mass that entrapped the nerve rootlets from L1 to the cauda equina and invaded the spinal cord from L4 to the cauda equina. Tissues were immersed in 10% neutral buffered formalin and routinely processed. Five micrometer paraffin-embedded sections were stained with hematoxylin and eosin (H&E) for routine histologic assessment. Immunohistochemistry was performed on sections of the spinal cord using canine leukocyte-specific antibodies for which the sources, reactivity, and staining protocol have been documented previously.2, 3 Histologically, dense sheets of pleomorphic, neoplastic round cells were present within the subarachnoid space of the spinal cord and brain, often obscuring the leptomeninges and multifocally invading the dura mater and subpial neuropil. The most extensive invasion involved the L4–6 and caudal spinal cord segments, in which neoplastic cells extended to and within the central canal. The neoplasm tapered from the thoracic to the cervical spinal cord, markedly expanded again around the medulla oblongata, cerebellum, and thalamus, and then tapered toward the cerebral hemispheres. It invaded along the subarachnoid space around the blood vessels within the superficial neuropil, outlined the cerebellar folia, and occasionally invaded the subpial neuropil. The affected nerve rootlets and the neuropil of the spinal cord and brain contained extensive edema and Wallerian degeneration. Consistent with the histiocytic origin demonstrated by immunocytochemistry of the CSF, neoplastic cells were broadly immunoreactive for CD18, inconsistently immunoreactive for CD45, and not immunoreactive for all other leukocyte antibodies (CD3, CD11d, CD79a, and pancytokeratin). A 9-year-old male castrated Pembroke Welsh Corgi was presented to the William R. Pritchard Veterinary Medical Teaching Hospital, University of California, Davis, for an acute onset of obtundation, head tilt, and difficulty in walking. The dog had received nonsteroidal anti-inflammatory treatment (unknown drug and dose) before presentation. Neurologic examination indicated moderate obtundation, tetraparesis, and mild generalized ataxia with a tendency to walk against the wall on the left side. Inconsistent menace response in the right eye, decreased response to nasal stimulation in the left naris, a mild head tilt to the right, and miotic pupils were noted. Proprioceptive positioning was evaluated by paw replacement reaction, and was absent in the right thoracic and pelvic limbs, and intact on the left. Neuroanatomic localization was consistent with multifocal brain disease. A CBC, serum biochemistry, urinalysis, thoracic radiographs, and abdominal ultrasound examination were unremarkable except for a mildly enlarged and heterogeneously echogenic right pancreatic limb interpreted as most consistent with previous pancreatitis. The dog was anesthetized and MR imaging of the brain was performed, including pre- and postcontrast T1W, T2W, proton density, and fluid-attenuated inversion recovery (FLAIR) images. A mass was identified, which appeared to arise from the lateral margin of the hippocampus, with an extensive component within the lateral ventricles bilaterally. The mass was a hypointense to gray matter on T1W images (Fig 6A) and isointense to slightly hyperintense on T2W images. On T2W images and FLAIR sequences, bilateral periventricular and peritumoral edema was observed (Fig 6B). The mass enhanced homogeneously with contrast administration (Fig 6C and D). (A) T1-weighted transverse magnetic resonance imaging (MRI) at the level of the zygomatic process of the temporal bone of dog 2. Note the bilateral hypointense mass associated with the lateral margin of the hippocampus. (B) Transverse fluid-attenuated inversion recovery image. Note the bilateral periventricular and peritumoral edema. Transverse (C) and dorsal plane (D) T1-weighted postcontrast images. Note the uniform, bilateral contrast enhancement of the mass with asymmetrical bilateral extension into the ventricular lamina. Cisternal CSF had a protein concentration of 135mg/dL (reference range, ≤ 25 mg/dL), 220 RBC/μL (reference range, 0 RBC/μL), and a markedly increased nucleated cell count of 1,550/μL (reference range, ≤ 3 cells/μL), with a differential cell count of 1% lymphocytes, 19% neutrophils, and 80% macrophages. On cytological evaluation, macrophages predominated, with nondegenerate neutrophils in lower numbers. A population of individual giant cells with rounded cytoplasmic margins was also present. These cells had a moderate to high N : C ratio and abundant, homogeneous, medium blue cytoplasm. Rare cells were erythrophagic. Nuclei were oval to reniform with lacy, open chromatin and were somewhat eccentrically placed within the cytoplasm. Binucleated and multinucleated cells were seen occasionally and featured marked anisocytosis and anisokaryosis. Numerous mitotic figures were noted within this population of large, immature round cells. The diagnosis was malignant neoplasia with associated marked mixed inflammation and mild prior hemorrhage. Additional CSF cytospin slides were made and assessed with antibodies against CD1c, CD11b, CD11c, cytokeratin (pancytokeratin and AE1/AE3), and GFAP along with a negative control. The neoplastic cells were not immunoreactive for cytokeratin or GFAP, moderately immunoreactive for CD1c, weakly to moderately immunoreactive for CD11c, and variably and weakly immunoreactive for CD11b. The diagnosis was histiocytic sarcoma with associated marked mixed inflammation. The dog recovered from anesthesia and was treated with an anti-inflammatory dosage of dexamethasone sodium phosphate (0.2 mg/kg/d) IV and lactated ringers solution with 20 mEq KCl/L was added IV (6.7 mL/kg/h). Twelve hours later, the dog was markedly obtunded. Because of rapid deterioration in neurological status and the poor prognosis associated with histiocytic sarcoma, the dog was euthanized. Clinically relevant gross necropsy findings were limited to the CNS. After fixation in 10% neutral buffered formalin, the brain was sectioned frontally and a soft tan mass that was bilaterally within the lateral ventricles and third ventricle was observed. It extensively invaded and effaced the dentate gyri and corpus callosum. Tissues were fixed in formalin, embedded in paraffin, sectioned at 5 μm, and stained with H&E. A section of the brain that included the dentate gyrus was immunohistochemically stained using a panel of leukocyte markers, as previously described in this report. On histopathologic examination, densely cellular sheets of neoplastic round cells, supported by moderate fibrovascular connective tissue, extensively effaced the dentate gyri and the corpus callosum. Few to numerous neoplastic round cells were scattered throughout the subarachnoid space and there was multifocal invasion of the subpial neuropil. The neoplastic cells were strongly immunoreactive with antibodies directed against CD45 and CD18. A subset of the neoplastic cell population, most notably within the meninges, was CD11d immunopositive. The neoplastic cell population exhibited no immunoreactivity with antibodies directed against CD3, CD79a, or CD20. Based on the cellular morphology and results of the immunohistochemical staining of the paraffin-embedded brain tissue, the mass was diagnosed as a histiocytic sarcoma. These results confirmed the diagnosis made antemortem by cytological and immunocytochemical evaluation of the CSF. The neoplasm was considered a primary CNS histiocytic sarcoma because there was no gross or histological evidence of neoplasia outside of the CNS. Histiocytic disorders are common in dogs and can be divided into reactive histiocytoses, cutaneous histiocytoma, localized histiocytic sarcoma, and disseminated histiocytic sarcoma (dHS)/malignant histiocytosis (mHS). dHS and mHS are histiocytic cell malignancies that either arise in 1 location with secondary widespread dissemination (dHS) or in many locations simultaneously (mHS).2, 3 These 2 diseases may be difficult to differentiate and, therefore, the terms frequently are used interchangeably. Dogs with dHS/mHS often have clinical signs of systemic disease because the internal organs are affected.3 Predilection sites include spleen, liver, lymph nodes, lungs, and bone marrow.4, 5 Bernese Mountain Dogs, Rottweilers, and Golden, Labrador, and Flatcoat Retrievers are predisposed to this disease,3, 6-18 whereas other breeds are affected sporadically.5, 19-22 Although many reports describe dHS/mHS affecting the CNS,4, 16, 20, 21 localized CNS histiocytic sarcoma has been infrequently reported.19, 20, 22 Three reports describe localized histiocytic sarcoma confined to the CNS of dogs, sparing other visceral organs,19, 20, 22 but bone marrow was not evaluated in these dogs. All 3 dogs were diagnosed with localized HS postmortem, 2 dogs had the CSF evaluated.20, 22 In 1 dog with diffuse leptomeningeal histiocytic sarcoma, the CSF nucleated cell count was 152 cells/μL, with a differential cell count of 50% neutrophils and 50% mononuclear cells and a total protein of 95 mg/dL. No atypical cells were recognized and CSF immunphenotyping was not performed.20 The other dog had a parenchymal brain mass and the cisternal CSF had a total nucleated cell count of 520 cells/μL, with a differential cell count of 7% neutrophils, 17% lymphocytes, and 76% monocytoid cells. The total protein (pyrogallol red methodology) was 51.8 mg/dL. The cytological interpretation was severe monocytoid—predominant, mixed cell pleocytosis, with cellular atypia suggestive of neoplasia.22 Postmortem, archived, unstained, air-dried, cytocentrifuged slides were immunophenotyped. The monocytoid cells were immunoreactive for CD1c and CD11d and were not immunoreactive for T and B cell markers (CD3 and CD79a). The CSF immunocytochemistry results supported the postmortem diagnosis of HS.22 The MR imaging findings were similar in both dogs reported here. In the first dog, the primary tumor was located in the L4–Cd segments whereas in the second dog, the primary tumor arose from the anatomical margins of the hippocampus with an extensive component in both lateral ventricles. In both cases, the tumors were contrast enhancing on T1W images, with diffuse meningeal enhancement in the area of the tumor as well as in other locations within the CNS. In the second dog, peritumoral edema was present. To our knowledge, this is the first report of antemortem diagnosis of primary CNS HS using CSF analysis. Similar to the dogs in previous reports,20, 22 the dogs in this report had marked, mixed, predominantly mononuclear pleocytosis with a moderate increase in the CSF protein concentration. CSF cytology warranted immunophenotyping because the neoplastic cell population was highly pleomorphic, exhibited both round to stellate and spindloid morphology, and was accompanied by a high number of inflammatory leukocytes, which complicated definitive interpretation without concurrent immunophenotyping. The large immature cells in the CSF were immunoreactive for CD1c and CD11c, variably and weakly immunoreactive for CD11b, but not immunoreactive for CD11d, CD3, or CD79a. The cell morphology and immunophenotype were indicative of histiocytic sarcoma of a dendritic cell origin.2, 3 In a report assessing histiocytic sarcomas of a dendritic cell origin in 39 dogs, the neoplastic cells were consistently immunoreactive for CD1c, CD11c, CD18, and CD45. The cell of origin in these tumors is believed to be an interstitial myeloid dendritic antigen-presenting cell. Postmortem immunohistochemistry performed on the CNS tissue from the dogs reported here confirmed the antemortem diagnosis. Neoplastic cells were strongly and diffusely immunoreactive for CD18, strongly and variably immunoreactive for CD45, and not immunoreactive for CD3, CD79a, and CD20. The MR image characteristics and CSF analysis, including immunocytochemical immunophenotyping and postmortem findings, confirmed the presence of a primary HS with metastasis within the CNS, possibly tracking along the meninges. Dogs with localized CNS HS may have a marked CSF pleocytosis with moderately increased protein concentration. When large, discrete atypical cells are present, immunophenotyping of CSF cells may be used to definitively confirm the histiocytic lineage of the atypical cells and hence diagnose HS. In the 2 dogs reported here, the MR findings of a primary tumor with diffuse T1W meningeal enhancement, CSF analysis, and CSF immunophenotyping permitted an antemortem diagnosis of HS, which was confirmed on postmortem evaluation. aGeneral Electric Signa LX, Milwaukee, WI bMagnevist (Gadopentate dimeglumine 469.01 mg/mL), Berlex Laboratories, Wayne, NJ

Background: Acute lymphoblastic leukemia (ALL) is a malignant neoplasia in which there is proliferation of lymphoid progenitor cells in the bone marrow, blood, and extramedullary sites. This disorder has a fast and progressive development; in dogs, cases of infiltration of ALL cells in the central nervous system (CNS) are uncommon and rare. Diagnosis can be achieved with the help of the clinical history and physical, radiographic, hematological, myelographic, and cerebrospinal fluid (CSF) tests in patients with or without neurological clinical signs. The present report aims to describe a case of ALL and the presence of lymphoblasts in the CSF of a dog with neurological clinical signs.Case: An 8-year-old Lhasa Apso dog was examined at the Veterinary Hospital of Universidade Federal do Paraná, Curitiba campus. At the physical examination, the animal exhibited apathy and paralysis of pelvic limbs, which progressed to tetraplegia. Abdominal palpation revealed presence of hepatosplenomegaly and absence of lymphadenomegaly. No alterations were observed in radiographs of the cervical, thoracic or lumbar spine. A complete blood count revealed presence of non-regenerative anemia (hematocrit = 22%), extreme lymphocytosis (185,229 cells/µL), lymphoblasts at a level of 72% (133,364 cells/µL), and thrombocytopenia (66,000 platelets/µL). The biochemical tests revealed increased alkaline phosphatase (859 IU/L). The levels of alanine aminotransferase, creatinine, urea, total protein, albumin, and globulin were normal. The diagnosis of ALL was achieved with the help of a myelogram. The myelogram findings included 39% of mature lymphocytes and 59% of lymphoblasts exhibiting large size, spherical shape, poorly delimited borders, with a high nucleus/cytoplasm ratio, marked cytoplasmic basophilia, and 2 to 3 evident nucleoli; metarubricytes (1%) and promyelocytes (0.6%) were also observed. The CSF contained an increased number of nucleated cells (27 cells/µL) comprising lymphocytes (43%), macrophages (33%), and segmented neutrophils (24%). Of the 11.6 lymphocytes per µL of CSF, 8.1 were lymphoblasts, which indicates infiltration of ALL cells in the CNS. The animal died one day after collection of bone barrow and CSF. Discussion: Relevant alterations observed in this case included the neurological signs caused by the infiltration of neoplastic cells in the CNS, severe leukocytosis and lymphocytosis, with large amounts of lymphoblasts in the blood and predominance of lymphoblasts in the bone marrow, which are alterations typically found in ALL. The animal also exhibited non-regenerative anemia and thrombocytopenia, which were secondary to infiltration of leukemic cells in the bone marrow. The CSF exhibited pleocytosis (27 cells/ µL), and 30% of the cells observed were lymphoblasts. Lymphoblast infiltration in the CNS of leukemic dogs is rare, and other studies have reported absence of neurological signs or neurological signs different from those observed in the present study. CSF analysis in indicated in cases of leukemia to assess leukemic cell infiltration in the CNS. In the case reported here, the plasma level of alkaline phosphatase was increased (859 IU/L) as a consequence of hepatomegaly and hepatic cholestasis. ALL is a very aggressive, proliferative neoplasia, and the resulting lymphoblasts infiltrated the CNS of the animal. In cases of ALL, performing complete blood count, myelogram, and CSF analysis is indicated whether the patients exhibit neurological signs or not.

High Levels of Nm23-H2 Protein Are a Specific and Invariable Feature of CML-CP Cells Due to Post Transcriptional Control Via a Bcr-Abl Dependent Pathway

IntroductionVisceral leishmaniasis (VL) is a potentially fatal parasitic infection occurring primarily in the tropics, subtropics, and southern Europe. A strong humoral response can be observed in VL due to polyclonal activation of B-lymphocytes, and it can also result in the production of rheumatoid factor (RF), anti-CCP and anti-nuclear antibodies. We present this case here of visceral leishmaniasis presenting as seropositive RA on the background of HIV and hepatitis B infection.Case descriptionWe present a case of a 46-year-old Caucasian male with a two-year history of skin nodules, symmetrical arthralgia and weight loss. He was diagnosed with HIV a decade ago which was well controlled with bictegravir, emtricitabine and tenofovir alafenamide. He received pulmonary tuberculosis (TB) treatment in 2012 and had chronic e-antigen-positive hepatitis B. He previously resided in Ukraine and travelled frequently to Crimea and the Middle East.There was no history of Raynaud’s, chest pain, shortness of breath, sicca symptoms, mouth ulcers, genital ulcers, hair loss or photosensitivity.On examination, he was cachectic with generalised lymphadenopathy and multiple subcutaneous (1-2cm, firm, mildly tender, non-ulcerating) forearm nodules. There was synovitis on two joints but no tenosynovitis or joint deformity. Initial investigations revealed pancytopenia, hyperuricemia, proteinuria, positive RF (>200 IU/ml) and anti-CCP, positive ANA and anti-Ro antibody and erosion of proximal phalanx of the big toe bilaterally. His CD4 count was 166 (33%) with undetectable HIV viral load. Imaging showed lymphadenopathy and splenomegaly (17.5cm).A diagnosis of rheumatoid arthritis with possible connective tissue disease overlap was made and he was initiated on hydroxychloroquine.Because of systemic symptoms and atypical skin nodules, further investigation was undertaken including lymph node, bone marrow and skin biopsies, all of which confirmed a diagnosis of leishmaniasis. Blood PCR was positive for L. donovani complex, and serological analysis showed direct agglutination test and k39 Ag positivity.Treatment was initiated by the infectious diseases team initially with amphotericin B 40 mg/kg monotherapy, later on further amphotericin B 30 mg/kg and miltefisone. He responded well as evidenced by reduced splenomegaly, resolution of skin nodules and improvement in cytopenia. His joint symptoms also improved except for the recurrence of ankle pain for which he received intra-articular steroids.DiscussionIt has been widely reported in literature that leishmaniasis can trigger autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosis. There have also been multiple studies confirming the presence of rheumatoid factor, anti-CCP and ANA in patients with leishmaniasis without any presence of symptomatic autoimmune disease. The mechanisms involved in the pathophysiology of autoantibody productions are not yet fully understood.There is some evidence that these are related to activation of polyclonal B cells due to a decreased regulatory T cell activity. Another possible mechanism would be production of autoantigen in response to cross reaction between the parasite and the hoarse tissue antigens. There have been multiple case reports of leishmaniasis in patients with rheumatoid arthritis who were taking anti-TNF biologics. This represents the reactivation of leishmaniasis due to immunosuppression. There has been a long-known link between HIV infection and leishmaniasis.Our patient was a known case of HIV. Interestingly, not only did he have significantly positive rheumatoid factor, anti-CCP, ANA and anti-Ro antibodies, but he also had active synovitis on his joints and even erosions were seen on the X-ray. He fulfilled the criteria for rheumatoid arthritis.Our case was unique in the sense that this is a case of visceral leishmaniasis in an HIV patient who presented with rheumatoid arthritis. Skin nodules in this patient were appearing similar to rheumatoid nodules. Later on, the biopsy confirmed that these were due to leishmaniasis. It is also a challenge to manage RA in patients with HIV especially when have already an opportunistic infection like leishmaniasis. A high prevalence of hyperuricemia in HIV infection has been reported in multiple studies. Our case did have raised urate but his disease was not consistent with gout clinically.Key learning points• This is a rare case showing seropositive erosive RA coexisting with VL in an HIV patient, underscoring the link between autoimmune diseases and chronic infections. Chronic infections are always considered an important differential in inflammatory arthritis or connective tissue disease especially when they present with systemic features of fever, weight loss and organomegaly.• Although this disease is considered rare in the Western hemisphere, in the diverse, multiethnic and immigrant UK population this should be considered an important differential diagnosis. Another key element in clenching these diagnoses is detailed travel history. Multidisciplinary approach is key in managing such difficult cases. Our case was managed jointly by the rheumatology, dermatology and infectious disease teams, and the national leishmaniasis multidisciplinary team from University College London Hospitals.• It is important to do a holistic assessment for these patients and get help from other specialities especially in such challenging cases.

A Comprehensive Study of Human Integrins in Pediatric Lymphoblastic Leukemia Supports a Role of CD49f (Integrin α6) in the Localization to Bone Marrow but Not Spinal Fluid

Background: Fungal infections affecting the central nervous system (CNS) represent a significant challenge in clinical practice due to their complexity and diversity of presentation. These infections generally affect immunocompromised patients. Early and accurate diagnosis is fundamental to defining treatment and improving the patient’s prognosis. Analysis of cerebrospinal fluid (CSF) is the method of first choice when there is a suspicion of fungal infection in the CNS, mainly because it is not very invasive and has high sensitivity. However, in more challenging cases, or where CSF analysis is inconclusive or cannot be performed, tissue biopsy is a viable alternative. Objective: To analyze data on cases of CNS fungal infection diagnosed by tissue biopsy. The evaluation will be carried out with data from a health service responsible for the pathology service of a local referral hospital, in the years 2022 to 2024. Methods: A descriptive analysis of biopsy results was carried out in order to draw up a diagnostic profile for fungal infections that affect the CNS using this method. These tests were provided by a pathology laboratory and comprise all the histological analyses from the neurosurgery department of a local referral hospital. The inclusion criteria were cases in which the patients had a suspected fungal infection of the CNS or in which the infectious diagnosis was incidental. Results: Between 2022 and 2024, the laboratory received 139 requests for analysis of neurological biopsies from the hospital. Of this total, around 4% were diagnosed with fungal infections, totaling 5 cases, 2 of which were cryptococci, and of these, 100% had another pathology as their suspected diagnosis. The following prevalence rates were also found: 35% of Gliomas (including glioblastomas), 20% of Meningiomas, 5% of carcinoma metastases, as well as other varied diagnoses such as Medulloblastoma, Hemangioblastoma, Ependymoma and Schwannoma. This distribution corroborates current literature, which indicates neurocryptococcosis as the most common fungal infection in the CNS, with an annual global incidence of 3.9 cases per 100,000 inhabitants. Conclusion: Fungal infections in the CNS are rare, but represent a significant diagnostic challenge. Tissue biopsy analysis, although less frequently used than CSF analysis, has proven to be a relevant tool in cases where the diagnosis is unclear or the CSF is inconclusive. This study highlights the need to consider fungal infections, such as neurocryptococcosis, in the differential diagnosis of CNS lesions, especially in immunocompromised patients or when symptoms do not correspond to more common pathologies. Accurate identification of these infections is vital for appropriate treatment and improving the patients prognosis.

Les sous-classes d’IgG dans le LCR présentent-elles un réel intérêt ?

BRAF-V600E in Peripheral Mononuclear Cells and Microglia-like Brain Cells Suggest Hematopoietic Origin of Langerhans Cell Histiocytosis-Associated Neurodegeneration

A 66-year-old woman was diagnosed in March 2007 with Bence-Jones protein-lambda positive (BJP k) multiple myeloma. After four cycles of bortezomib and dexamethasone, complete response was achieved in 2009. Radiotherapy was performed for skull and spine lesions in 2010 and 2011. She remained in stable disease with disappearance of M protein and bone marrow plasma cells on maintenance therapy with bortezomib, thalidomide, and dexamethasone. However, in May 2012, serum LDH began to elevate and multiple subcutaneous plasmacytoma appeared. Subcutaneous tumors increased following administration of lenalidomide and dexamethasone. Bone marrow examination in September 2012 demonstrated 50.8 % plasma cells. Cytogenetic analysis revealed a complex karyotype including chromosome 13 deletion and t(8;14)(q24;q32) in 19 of 20 metaphase cells. Although VCAP (vincristine, cyclophosphamide, doxorubicin, and prednisolone) slightly decreased subcutaneous tumors, she complained of headache, pain in the neck, dizziness, nausea, and vomiting in February 2013. Laboratory studies showed serum protein 6.2 g/dL, LDH 711 IU/L (normal range 80–200 IU/L), b2 microglobulin 2.5 mg/L, free light chain (FLC) j\ 0.6 mg/L (3.3–19.4 mg/L), k 269.0 mg/L (5.7–26.3 mg/L), and urine M protein 118 mg/24 h. Brain magnetic resonance imaging demonstrated meningeal contrast enhancement, particularly in the posterior fossa (Fig. 1). A lumbar puncture revealed a cerebrospinal fluid (CSF) protein of 48 mg/dL (10–53 mg/dL) and a marked pleocytosis of 300 cells/lL. The CSF cytology showed numerous plasma cells with prominent nucleoli (Fig. 2). FLC assay of the CSF demonstrated k chain restriction: j\ 0.6 mg/L, and k 435.0 mg/L. A diagnosis of meningeal myelomatosis was made. No plasma cells were identified in the peripheral blood. The patient received intrathecal chemotherapy of methotrexate and dexamethasone with no response, and died 1 month after the diagnosis. Meningeal myelomatosis, defined by the presence of monoclonal plasma cells in the CSF, is extremely rare and accounts for approximately 1 % of myeloma patients. Previous reports suggest an association of meningeal myelomatosis with extramedullary disease, plasmablastic morphology, cytogenetic abnormalities, high LDH levels, and circulating plasma cells. These are characteristics of aggressive myeloma, some of which we confirmed in our patient. Meningeal myelomatosis occurs as a terminal event in the majority of patients. The prognosis remains poor despite aggressive local and systemic treatment, including stem cell transplantation, with a median overall survival of only a few months. Recently, investigators have showed that the novel agents, thalidomide and lenalidomide, cross the blood–brain barrier and are effective for the management of meningeal myelomatosis [1–3]. However, our patient developed meningeal myelomatosis after receiving these same agents. Cerebral symptoms are commonly seen in terminal-stage myeloma patients from hypercalcemia, uremia, hyperviscosity syndrome, or toxicity of treatment. Physicians should be alert to the possibility of meningeal myelomatosis. The FLC assay of CSF will be helpful for diagnosis even when the CSF cell count and protein level are not elevated. To our knowledge, this is the first report of the use of FLC assay of CSF for diagnosis of meningeal myelomatosis. M. Ise (&) C. Sakai K. Kumagai Department of Hematology-Oncology, Chiba Cancer Center, 666-2 Nitona-cho, Chuo-ku, Chiba, Chiba 260-8717, Japan e-mail: mise@chiba-cc.jp

Combination therapy of acute promyelocytic leukaemia (APL) with chemotherapy and all-trans retinoic acid (ATRA) significantly reduces the incidence of relapse. The majority of relapses occur in the bone marrow; however, recent reports suggest an increasing incidence of central nervous system (CNS) relapse, which was rarely reported prior to the widespread use of ATRA (Currie et al, 1997; Evans et al, 1997; Anguita et al, 1999). It has been postulated that ATRA increases the expression of adhesion molecules on leukaemic promyelocytes and their ligands on endothelial cells, thereby increasing their migratory capacity and predisposing to extramedullary leukaemia (Evans et al, 1997). We describe a further case of APL relapsing in the CNS and discuss other possible aetiological factors. A 20-year-old man with hypergranular APL and documented t(15;17) presented with hemiparesis. A computerized tomography (CT) brain scan showed intracerebral haemorrhage, and the white blood cell (WBC) count was 2·3 × 109/l. He was treated with ATRA 45 mg/m2/d, and complete morphological and cytogenetic remission was confirmed on d 40. Four cycles of anthracycline-only consolidation were given according to a modified regimen based on the protocol used by the PETHEMA (Programme for the Study and Treatment of Haematological Malignancies) group (Sanz et al, 2000). This comprised daunorubicin 45 mg/m2 for 3 d for two cycles, idarubicin 10 mg/m2 for 3 d, and mitoxantrone 10 mg/m2 for 3 d. After consolidation the bone marrow was negative for the PML-RARα rearrangement using reverse transcription-polymerase chain reaction (RT-PCR) (sensitivity 10−4). He received maintenance therapy with daily 6-mercaptopurine 100 mg, weekly methotrexate 10 mg/m2 and intermittent courses of ATRA 45 mg/m2/d for 15 d every 3 months. Three months later he developed increasing hemiparesis and headache. A CT brain scan showed an old parietal infarct. Numerous hypergranular promyelocytes were seen on cerebrospinal fluid (CSF) examination, consistent with CNS relapse of APL. Bone marrow examination showed complete morphological and molecular remission. ATRA was restarted and he received intrathecal methotrexate alternating with cytosine arabinoside (ara-C), followed by cranial radiotherapy. Seven months later CSF examination is normal and he remains in morphological remission. The GIMEMA (Gruppo Italiano Malattie Emetologiche dell'Adulto) group recently reported a significantly higher proportion of relapses in the CNS when ATRA was added to idarubicin for induction in APL (Specchia, 1999). Although modulation of adhesion molecules by ATRA may promote entry of leukaemic promyelocytes into the CSF, there may be alternative explanations (Evans et al, 1997). Cerebral bleeding may seed the CSF with leukaemic promyelocytes which are then relatively protected from systemic chemotherapy. This may contribute to later CNS relapse in the presence of bone marrow remission. CNS relapse with early marrow relapse 2 months after consolidation has been reported previously in a man with cerebral haemorrhage at first diagnosis (Currie et al, 1997). It is possible that subclinical cerebral bleeding prior to diagnosis seeded the CSF with leukaemia in other reported cases. This is consistent with a recent report demonstrating an increased incidence of CNS relapse in children with acute lymphoblastic leukaemia (ALL) in whom diagnostic lumbar puncture was traumatic (Gajjar et al, 2000). As the prognosis of APL improves, CNS relapses of APL may be increasingly recognized. Anthracycline-only consolidation has not been associated with reduced antileukaemic efficacy (Sanz et al, 2000). However, ara-C may have a role in treating subclinical CNS leukaemia and a similar case of CNS relapse while in morphological and molecular remission has been reported following anthracycline-only consolidation (Anguita et al, 1999). Therefore anthracycline-only consolidation may not eradicate subclinical CNS leukaemia and its wider use may be associated with an increased incidence of CNS relapses. Furthermore, our case suggests that CNS prophylaxis should be considered for patients who first present with CNS bleeding and receive anthracycline-only consolidation. We would like to acknowledge the role of Dr J. Treleaven, Royal Marsden Hospital, in the initial diagnosis and management of this case and thank her for referring the patient to our department.

Use Of Rearranged Immune Receptor Sequencing To Measure Minimal Residual Disease (MRD) In Bone Marrow, Cerebrospinal Fluid and Testes In Relapsed Childhood Acute Lymphoblastic Leukemia

Ig-secreting cells pass the blood-brain barrier: studies on κ and λ light chain secreting cells in plasma cell dyscrasia

Multicolor flow cytometric analysis of the CD45 antigen provides improved lymphoid cell discrimination in bone marrow and tissue biopsies

Simple SummaryThe invasive nature of surgical biopsies prevents their sequential application to monitor disease. Single biopsies fail to reflect cancer dynamics, intratumor heterogeneity, and drug sensitivities that change over time. Detection and characterization of cell-free circulating tumor DNA in biofluids from patients with solid tumors may better support disease monitoring and provide advanced molecular information for clinical decision-making toward personalized medicine. Here, we investigated the cell-free DNA characteristics in blood, bone marrow, cerebrospinal fluid, and urine provided from 84 infants and children with low-, intermediate-, or high-risk neuroblastoma. We report characteristic size distribution and concentration patterns for each biofluid to provide information to support the development of successful liquid biopsy biobanking strategies. We investigate potential correlations between disease activity and cfDNA concentration and provide strong evidence that markers specific for neuroblastoma can be detected in very small blood volumes from infants.Liquid biopsy strategies in pediatric patients are challenging due to low body weight. This study investigated cfDNA size distribution and concentration in blood, bone marrow, cerebrospinal fluid, and urine from 84 patients with neuroblastoma classified as low (n = 28), intermediate (n = 6), or high risk (n = 50) to provide key data for liquid biopsy biobanking strategies. The average volume of blood and bone marrow plasma provided ranged between 1 and 2 mL. Analysis of 637 DNA electropherograms obtained by Agilent TapeStation measurement revealed five different major profiles and characteristic DNA size distribution patterns for each of the biofluids. The proportion of samples containing primarily cfDNA was, at 85.5%, the highest for blood plasma. The median cfDNA concentration amounted to 6.28 ng/mL (blood plasma), 58.2 ng/mL (bone marrow plasma), 0.08 ng/mL (cerebrospinal fluid), and 0.49 ng/mL (urine) in samples. Meta-analysis of the dataset demonstrated that multiple cfDNA-based assays employing the same biofluid sample optimally require sampling volumes of 1 mL for blood and bone marrow plasma, 2 mL for cerebrospinal fluid, and as large as possible for urine samples. A favorable response to treatment was associated with a rapid decrease in blood-based cfDNA concentration in patients with high-risk neuroblastoma. Blood-based cfDNA concentration was not sufficient as a single parameter to indicate high-risk disease recurrence. We provide proof of concept that monitoring neuroblastoma-specific markers in very small blood volumes from infants is feasible.

In a patient with precursor B-cell acute lymphoblastic leukemia (ALL) associated with eosinophilia that completely responded to induction chemotherapy, we assayed serial remission cerebrospinal fluid and bone marrow specimens for minimal residual disease using a quantitative polymerase chain reaction assay to assess for clone-specific immunoglobulin heavy-chain gene cluster (IGH) gene rearrangement. Cerebrospinal fluid eosinophilia and minimal residual disease were detected on day 406, preceding the morphologic diagnosis of central nervous system relapse on day 578. By day 841, the bone marrow had 35% blasts. Despite aggressive therapy, including unrelated umbilical cord blood transplantation, the patient developed testicular and bone marrow relapses and died of disease. We conclude that increasing levels of minimal residual disease in cerebrospinal fluid can predict recurrence of ALL prior to clinical and morphologic relapse. Furthermore, we demonstrate a novel translocation in this tumor, the t(5;9)(q31;p24), that possibly led to fusion of the interleukin-3 (IL3) (5q31) and JAK2 (9p24) genes and may explain the concomitant appearance of eosinophilia and ALL.