Cerebrospinal Fluid Sediment Research Articles

Laboratory diagnostics of Acanthamoeba spp. infections

Protozoa <i>Acanthamoeba</i> castellanii are single-celled, free-living, cosmopolitan amoebae that enter the body mainly through the mucous membrane of the mouth, nasal cavity, cornea, respiratory system, damaged skin and intestinal mucosa. In addition, they can be vectors for microorganisms such as bacteria, viruses, fungi and protozoa. In the life cycle A. castellanii may take the form of trophozoites and cyst tenacity to environmental conditions. This amoeba is an etiologic agent of dangerous human diseases: <i>Acanthamoeba</i> keratitis (AK), granulomatous amoebic encephalitis (GAE), pneumonia or changes in other organs, such as the liver, kidneys and skin. The aim of the article is to present current knowledge about laboratory diagnosis of <i>Acanthamoeba</i> spp. infection, which is based on the search for trophozoites and cysts in the material collected from the patient. In the case of suspected GAE, it is also advisable to study cerebrospinal fluid sediment and perform imaging tests such as computed tomography or magnetic resonance imaging. In the case of <i>Acanthamoeba</i> keratitis, an amoeba culture (obtained from a biopsy or corneal scrapings) is established in vitro, and then identified by light microscopy. The methods of molecular biology are also useful in detecting, identifying, and determining the potential pathogenic abilities of the amoebas. After the detection of <i>Acanthamoeba</i> spp., it is important to differentiate pathogenic isolates from non-pathogenic ones. From a clinical point of view, to diagnose <i>Acanthamoeba</i> spp. infection it is necessary only to identify the type of organism and to determine the pathogenicity of the isolate.

Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis

Tuberculous meningitis (TBM) is one of those most serious manifestations of extra-pulmonary tuberculosis and prompt diagnosis and treatment is required for better clinical outcome. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the Multiplex polymerase chain reaction (PCR) technique, using primers directed against the insertion sequence IS6110 and MPB64 gene for the detection of Mycobacterium tuberculosis in Cerebrospinal fluid (CSF), for rapid diagnosis of TBM patients. 102 CSF samples were analyzed from patients suspected with TBM along with a control group of 10 patients having other neurological disorders. CSF sediments were analyzed individually for M.tuberculosis DNA by Multiplex PCR using two set of primers targeting insertion sequence IS6110 and gene MBp64, which is very specific for MTBC. Out of 37 patients diagnosed with TBM clinically, MPB64 PCR was positive in 22, IS6110 PCR was positive in 28, both PCR using Multiplex were positive in 34 and Microscopy was positive in one. Thus Sensitivity of MPB64 PCR, IS6110 PCR, Multiplex PCR and Microscopy were found to be 62.3%, 75.4%, 91.8% and 2.7% respectively. In non TBM group PCR was negative in all cases hence, the specificity was 100%. Multiplex PCR system using primers targeting IS6110 and MPB64, for the detection of M.tuberculosis DNA in CSF samples, has high sensitivity than any one of them alone, and could be used for the early detection of TBM in CSF samples.

Successful Treatment of Granulomatous Amoebic Encephalitis with Combination Antimicrobial Therapy

Granulomatous amoebic encephalitis (GAE) is a rare but fatal infection. Due to its nonspecific symptoms and laboratory and neuroradiological findings, it is rarely diagnosed antemortem. We herein present the case of a 72-year-old Japanese woman who was diagnosed with GAE following the detection of a pathogen similar to Balamuthia mandrillaris under a microscopic examination of cerebrospinal fluid sediment and who achieved remission with combination antimicrobial therapy. There are no previous reports of pathogens similar to B. mandrillaris being detected in cerebrospinal fluid antemortem; therefore, this case may be used as a benchmark for further studies.

Improved Models of Mini Anion Exchange Centrifugation Technique (mAECT) and Modified Single Centrifugation (MSC) for Sleeping Sickness Diagnosis and Staging

Improved Models of Mini Anion Exchange Centrifugation Technique (mAECT) and Modified Single Centrifugation (MSC) for Sleeping Sickness Diagnosis and Staging

Late Cerebral Graft versus Host Reaction in a Bone Marrow Transplanted Girl with Hurler (MPS I) Disease

A girl with Hurler disease (MPS IH) underwent allogeneic stem cell transplantation at 13 months of age with her one HLA-B antigen mismatch mother as donor. The procedure was complicated by cerebral hemorrhage and a ventricular-peritoneal shunt device was inserted. Mild GVH reactions were rapidly reversed. One year after transplantation ventriculitis was suspected and the shunt was replaced by a ventricular drainage catheter. Antibiotics had no effect and graft-versus-host disease (GVHD) was diagnosed. All symptoms were reversed by prednisolone and cyclosporine. Increased albumin and pleocytosis in the cerebrospinal fluid (CSF) normalized concomitantly. Electron microscopy of the CSF sediment showed debris consisting of numerous complex aggregates of thin lamellae and electron dense fragments with a tight lamellar texture. Biochemical analysis of the CSF sediment proved that the debris contained galactosylceramide and sulfatide. The electron microscopic and biochemical findings were interpreted to represent stripping of central myelin as a result of subacute GVHD in the central nervous system and its desquamation from the brain parenchyma into the ventricular CSF through the post-hemorrhage defect. From reversal of the GVHD at 2 years of age until follow-up at 10 years of age the clinical condition remained stable with no recurrence or deterioration.

Detection of Chlamydial Bodies and Antigens in the Central Nervous System of Patients with Multiple Sclerosis

To examine a possible relationship between Chlamydia pneumoniae infection and multiple sclerosis (MS), we undertook an immunohistochemical (IHC), molecular, and ultrastructural comparison of central nervous system (CNS) tissue and cerebrospinal fluid (CSF) sediment from patients with MS and control individuals with other neurological diseases (ONDs). In 7 of 20 MS cases, IHC staining was seen in association with ependymal surfaces and periventricular regions of formalin-fixed brain tissue, by use of 3 different antichlamydial antibodies. There was no staining with any of the 3 antichlamydial antibodies in formalin-fixed brain tissue from OND controls (n=17). With available frozen CNS tissue, polymerase chain reaction (PCR) studies for the presence of C. pneumoniae genes were performed. The presence of a PCR signal was confirmed in 5 of 8 MS cases and in 3 of 18 OND controls. In an examination of CSF sediment by electron microscopy, we observed electron-dense structures resembling chlamydial organisms in CSF sediments from 11 of 20 MS cases and 2 of 12 OND controls. The presence of immunogold-labeled electron-dense bodies was correlated with the presence of a PCR signal in 10 of 11 MS cases. Results of studies using these different approaches support our suspicion of the presence of chlamydial organisms in the CNS, in a subset of patients with MS.

Genomic variation in the PLCD gene region of clinical strains of Mycobacterium Tuberculosis

Tuberculous meningitis (TBM) is one of those most serious manifestations of extra-pulmonary tuberculosis and prompt diagnosis and treatment is required for better clinical outcome. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the Multiplex polymerase chain reaction (PCR) technique, using primers directed against the insertion sequence IS6110 and MPB64 gene for the detection of Mycobacterium tuberculosis in Cerebrospinal fluid (CSF), for rapid diagnosis of TBM patients.102 CSF samples were analyzed from patients suspected with TBM along with a control group of 10 patients having other neurological disorders. CSF sediments were analyzed individually for M. tuberculosis DNA by Multiplex PCR using two set of primers targeting insertion sequence IS6110 and gene MBp64, which is very specific for MTBC.Out of 37 patients diagnosed with TBM clinically, MPB64 PCR was positive in 22, IS6110 PCR was positive in 28, both PCR using Multiplex were positive in 34 and Microscopy was positive in one. Thus Sensitivity of MPB64 PCR, IS6110 PCR, Multiplex PCR and Microscopy were found to be 62.3%, 75.4%, 91.8% and 2.7% respectively. In non TBM group PCR was negative in all cases hence, the specificity was 100%.Multiplex PCR system using primers targeting IS6110 and MPB64, for the detection of M. tuberculosis DNA in CSF samples, has high sensitivity than any one of them alone, and could be used for the early detection of TBM in CSF samples.

Guillain-Barré syndrome with marked pleocytosis or a significant proportion of polymorphonuclear granulocytes in the cerebrospinal fluid: neuropathological investigation of five cases and review of differential diagnoses.

In cases with otherwise clinically typical Guillain-Barré syndrome (GBS), pronounced cerebrospinal fluid (CSF) pleocytosis or the mere presence of CSF-polymorphonuclear granulocytes should alert the physician to consider alternative diagnoses. Therefore, we retrospectively studied the neuropathology of central and peripheral nervous system in two cases with a CSF cell count of more than 50/microl and in three cases with a significant proportion of polymorphonuclear granulocytes in the CSF sediment. All cases fulfilled the required criteria for the diagnosis of GBS, the duration from onset to death ranged from 4 to 100 days. Neuropathological investigations included routine staining procedures and immunohistochemistry for antigens of glial and haematopoetic cells as well as for products of relevant neurotropic viruses. Demyelinating polyradiculitis was present in four cases, in one patient with a survival time of 4 days the type of damage to myelinated fibres was unclassifiable. In the central nervous system a consistent finding was diffuse activation of microglia, only one case showed mild meningeal and lower brainstem inflammation. Viral products were generally absent. In summary, the neuropathological findings confirm that marked CSF pleocytosis or the presence of polymorphonuclear granulocytes does not rule out the diagnosis of GBS.

Acanthamoeba spp. as agents of disease in humans.

Acanthamoeba spp. are free-living amebae that inhabit a variety of air, soil, and water environments. However, these amebae can also act as opportunistic as well as nonopportunistic pathogens. They are the causative agents of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis. Immuno compromised individuals, including AIDS patients, are particularly susceptible to infections with Acanthamoeba. The immune defense mechanisms that operate against Acanthamoeba have not been well characterized, but it has been proposed that both innate and acquired immunity play a role. The ameba's life cycle includes an active feeding trophozoite stage and a dormant cyst stage. Trophozoites feed on bacteria, yeast, and algae. However, both trophozoites and cysts can retain viable bacteria and may serve as reservoirs for bacteria with human pathogenic potential. Diagnosis of infection includes direct microscopy of wet mounts of cerebrospinal fluid or stained smears of cerebrospinal fluid sediment, light or electron microscopy of tissues, in vitro cultivation of Acanthamoeba, and histological assessment of frozen or paraffin-embedded sections of brain or cutaneous lesion biopsy material. Immunocytochemistry, chemifluorescent dye staining, PCR, and analysis of DNA sequence variation also have been employed for laboratory diagnosis. Treatment of Acanthamoeba infections has met with mixed results. However, chlorhexidine gluconate, alone or in combination with propamidene isethionate, is effective in some patients. Furthermore, effective treatment is complicated since patients may present with underlying disease and Acanthamoeba infection may not be recognized. Since an increase in the number of cases of Acanthamoeba infections has occurred worldwide, these protozoa have become increasingly important as agents of human disease.

Rapid diagnosis of cryptococcal meningitis by microscopic examination of centrifuged cerebrospinal fluid sediment.

The classic India ink test is positive in only half of cryptococcal meningitis cases, and reliable, rapid cryptococcal antigen (CRAG) testing requires technical expertise and facilities not always available. We therefore examined cerebrospinal fluid (CSF) sediment using May-Giemsa, periodic acid-Schiff, and Gram stains in 16 patients with cryptococcal meningitis. The India ink test was positive in seven patients (44%), while microscopic examination of sediment revealed cryptococci in 13 (81%); in six of these 13 the India ink test was negative. Both methods failed to detect the pathogen in the remaining three patients. CRAG testing in CSF was negative in two patients (one with acquired immunodeficiency syndrome, one with diabetes mellitus) whose India ink test also was negative while cryptococci were identified in their CSF sediment. No false positives occurred with CSF May-Giemsa staining in 27 cases of aseptic meningitis with negative cultures for Cryptococcus. In all, microscopic examination of centrifuged and stained CSF sediment proved more sensitive for rapid diagnosis of cryptococcal meningitis than the India ink method, and in two of our patients cryptococci were seen in centrifuged CSF sediment despite negative CRAG and India ink tests.

Complement-derived polypeptide C3adesArg as a mediator of inflammation at the blood-brain barrier in a new experimental cat model.

The effect of the complement-derived polypeptide C3adesArg as a mediator of inflammation in the central nervous system was examined. Twenty-five anesthetized cats received 4 mg of this polypeptide by intraventricular injection, 20 cats who served as controls received saline. Cerebrospinal fluid (CSF) was sampled 3 h after intraventricular injection and the brains were removed. For assessment of the permeability of the blood-brain barrier the CSF penetration of four antibiotics, which were given intravenously was measured. Five control animals were employed for each antibiotic (tobramycin, ampicillin, imipenem, fosfomycin), whereas six C3adesArg-treated animals were used for each antibiotic and seven for tobramycin. Besides CSF levels of glucose, the prostanoids 6-keto-prostaglandin F1 alpha, thromboxane B2 and prostaglandin E2 were measured. The morphological examinations in the CSF sediments and histological brain sections in the C3adesArg-treated animals disclosed a distinct inflammation with leptomeningeal and perivascular infiltration of polymorphonuclear granulocytes compared to normal findings in the controls. The CSF/serum ratios of all of the antibiotics were markedly elevated compared to controls, indicating a blood-brain barrier disruption. The levels of all prostanoids were significantly higher in the treatment group than in the control group, whereas the glucose levels were lower. These findings are in accordance with a granulocytic meningitis as seen in some infections at the acute stage. It is concluded that C3adesArg acts as a mediator of inflammation in the central nervous system.

The clinical significance of streptococcal species isolated from cerebrospinal fluid.

Alpha-hemolytic (viridans) streptococci are often isolated from cerebrospinal fluid (CSF); however, the significance of such isolates is poorly understood. In order to clarify the clinical significance of isolating these organisms from CSF, we did a retrospective analysis of 43 patients, from whom eight different species of alpha-hemolytic streptococci were recovered. Eight patients (19%) had significant infections based on bacteriologic, laboratory, and clinical findings. Significant infections were associated with S. sanguis, S. salivarius, S. intermedius, S. faecalis, and S. bovis. Thirty-five patient isolates (81%) from CSF were considered as contaminants, with S. mitis being the most frequently isolated organism (49%). Direct gram stain of CSF sediment, CSF glucose concentration, and CSF cell differential were clearly abnormal in most patients with significant infections, in contrast to patients with streptococci isolated as contaminants. Cultures of the lumbar puncture skin site yielded streptococci and other bacteria, suggesting a possible reservoir for contaminants.

Electron microscopic studies of cerebrospinal fluid sediment in demyelinating disease.

Cerebrospinal fluid specimens from 31 patients with multiple sclerosis (MS) and 2 with progressive multifocal leukoencephalopathy (PML) were subjected to ultracentrifugation, and the resulting pellets were examined in an electron microscope. Cell types seen in the pellets included lymphocytes, occasional plasma cells, polymorphonuclear leukocytes, monocytes, eosinophils, lipid-laden macrophages, and fibroblasts. The most interesting noncellular elements were extracellular myelin fragments, recognizable by their characteristic alternation of major dense lines and intraperiod lines. Myelin fragments were seen in the CSF from 7 of 9 patients with MS in exacerbation involving areas other than the optic nerve. These fragments were not observed in 4 specimens from patients with acute attacks manifested by optic neuritis. Myelin fragments were present in 1 of the 2 patients with PML. These observations indicate that a portion of the myelin destruction seen in MS and PML occurs extracellularly, with release of myelin fragments and degradation products into the CSF.

Ependymitis in mumps virus meningitis. Electron microscopical studies of cerebrospinal fluid.

Cerebrospinal fluid (CSF) sediment from six patients with mumps meningitis was examined with the electron microscope and the findings compared with those in six other cases of presumed viral meningitis or encephalitis. Ependymal cells and cytoplasmic inclusions of viral nucleocapsid-like material were found in the CSF of each of the six patients with mumps. Ependymal cells were found in only one of the CSF sediments from the other patients, and no viral inclusions were found. These findings indicate that ependymitis regularly occurs in mumps meningitis. This supports the hypothesis that mumps virus may be a cause of aqueductal stenosis and hydrocephalus in man.

Diagnosis of meningeal leukemia in acute childhood lymphocytic leukemia with periodic acid-schiff reaction of cytocentrifuged liquor.

The cerebrospinal fluid (CSF) of 22 consecutive untreated children with acute lymphocytic leukemia who entered total study VII from September to December 1971 at St. Jude Children’s Research Hospital was examined. Four patients had periodic acid-Schiff (PAS)-positive, Sudan black B- and peroxidase-negative lymphoblasts in the CSF at diagnosis. Two of the 4 developed meningeal leukemia 3 and 5 months following diagnosis. None revealed pleocytosis and/or detectable atypical cells on a Wright-stained smear of CSF sediment at diagnosis. Two patients had no more detectable PAS-positive lymphoblasts after ‘prophylactic’ central nervous system (CNS) radiotherapy. They are in continuous complete remission for 9 and 10 months. The recognition of a small number of CSF-abnormal cells with PAS reaction and the differentiation of blasts from inflammatory cells may allow an earlier diagnosis and treatment of CNS involvement in childhood acute lymphocytic leukemia.

A simplified procedure for spinal fluid cytology.

EXAMINATION of the cells in cerebrospinal fluid (CSF) often provides important evidence of the nature and course of disease of the nervous system. Yet, the methods most frequently used for microscopic examination are generally unsatisfactory. The morphologic features of cells are inadequately shown in a counting chamber. Stained smears of the centrifuged CSF sediment, while affording better cytologic definition, may result in severe distortion of cells. In addition, some of the cells may be lost. In an attempt to overcome these difficulties, a variety of other procedures for collection of CSF have been devised. Sedimentation of CSF specimens using a variety of chambers1,2provides superior preservation of cytologic detail, but is time consuming, fails to afford a complete collection of cells, and requires special equipment not readily available. The use of porous, cellulose, plastic filters (Millipore) to collect cells for examination3has become a standard technique in

Intrathecal arabinosyl cytosine in meningeal leukemia

Intrathecal arabinosyl cytosine was administered on a twice weekly schedule, in a dosage range of 5 to 70 mg per square meter of body surface area, to 13 children with meningeal leukemia. Eleven children with acute lymphocytic leukemia had been previously treated with intrathecal methotrexate, and 7 had received cranial or craniospinal radiotherapy. Seven of these 11 children responded with relief of symptoms and reduction of cerebrospinal fluid (CSF) cell count to less than 10 mononuclear cells per mm3 of CSF and less than 2 leukemic cells on one smear of CSF sediment. The median duration of response was 28 days. Vomiting occurred in 5 children after treatment, headache in 2, and fever in one. Neither leukopenia nor other systemic toxicity was observed. It is concluded that intrathecal arabinosyl cytosine has therapeutic activity against meningeal leukemia.

THE DIAGNOSIS OF METACHROMATIC LEUCODYSTROPHY DURING LIFE.

The authors report the study of saliva sediment in 4 cases of juvenile metachromatic leucodystrophy belonging to the same family (and else in a sister of one of these cases presenting the characteristic neurological picture but with no metachromasia demonstrable by the Austin test in urine or by biopsies), in 6 normal relatives of the patients with Scholz disease, in 9 cases of various diseases of the nervous system, and in 10 normal subjects. The presence of metachromatic bodies staining in a pinkish red colour with acid blue toluidine dye was demonstrated in the saliva sediment of the 4 cases of metachromatic leucodystrophy. In 2 of these patients biopsies of the parotid gland, stained with cresyl violet dye, showed the presence of intracellular brownish metachromatic bodies. In these 2 cases the study of cerebrospinal fluid sediment also disclosed the presence of metachromatic bodies. Furthermore, a chromatographic qualitative test for metachromatic lipids yielded positive results in saliva, cerebrospinal fluid, and urine sediments. The conclusion was drawn that the search for metachromatic bodies in cerebrospinal fluid and mainly in saliva sediment may be of help in disclosing or ratifying the diagnosis of metachromatic leucodystrophy during life.

IMMUNOFLUORESCENT TECHNIQUES IN BACTERIAL MENINGITIS. IDENTIFICATION OF NEISSERIA MENINGITIDIS AND HEMOPHILUS INFLUENZAE.

Purulent meningitis is a medical emergency in which prompt and accurate diagnosis and rapid institution of appropriate antibacterial therapy are essential to preserve life and to prevent serious and disabling sequelae. Microscopic examination of the stained cerebrospinal fluid sediment is a key to the correct, rapid identification of the microorganism responsible for the meningitis. This initial impression is confirmed when the organism noted on slides can be cultured and identified with greater precision. In clinical practice, however, these two examinations are sometimes neither satisfactory nor conclusive. Haggerty 1 has pointed out that even in experienced hands a 10% error is inherent in examination of the Gram-stained spinal fluid smear; additionally, no organism is seen on smear but the culture will yield a causative organism in 25% of cases; in 15% no organism is identified on smear or on culture. Meningococci are very delicate organisms and their survival can be jeopardized

The clinical spectrum of bacterial meningitis

E PIDEMIC meningitis with a rash of “purple spots” was clearly described by Vieusseux following an outbreak in Geneva in 1805 [7]. In the era before diagnostic bacteriology, meningitis was known to be an occasional complication of pneumonia and tuberculosis. In 1886 Frankel as well as Foa and Bondoni-Uffreduzzi demonstrated the pneumococcus in human spinal fluid. The following year Weichselbaum noted gram-negative diplococci in six cases of acute meningitis. Hemophilus influenza meningitis was described by Slawyk in 1899 [Z]. During the ensuing thirty years many of the less common etiologic agents were isolated, including the staphylococcus and a number of coliform organisms. More recently Mima polymorpha [3-51 and Listeria monocytogenes [6,7] have received attention as occasional causes of bacterial meningitis. logic diagnosis was established by culture of the cerebrospinal fluid. (Table I.) In eight cases the causative organism grew in blood cultures and was seen in the gram stain of the cerebrospinal fluid sediment. In six cases the diagnosis of meningococcal meningitis was made in patients with a purpuric rash and sterile cerebrospinal fluid containing typical gram-negative diplococci. In twenty-two cases the etiologic agent was unknown. In twelve of these, definite organisms were seen in the sediment of the cerebrospinal fluid. In two additional cases the diagnosis of purulent meningitis was confirmed at autopsy. The remaining patients recovered promptly from a clinically typical illness; in each instance the sugar in the cerebrospinal fluid was less than