Alberto Juan Dorta-Contreras

Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients

BackgroundToxoplasmic encephalitis (TE) is one of the most common opportunistic infections in immunocompromised patients. In Cuba, despite the highly active antiretroviral therapy, TE is still the most important cause of cerebral mass lesions in patients infected with the human immunodeficiency virus (HIV). The detection of Toxoplasma gondii by PCR may facilitate the diagnosis and follow-up of TE in acquired immunodeficiency syndrome (AIDS) patients by direct identification of parasite DNA in clinical samples. The aim of the present study was to evaluate a rapid PCR method using the B1 gene to detect T. gondii in cerebrospinal fluid (CSF) samples from patients with suspected TE.MethodsCSF samples from AIDS and HIV-negative patients were analyzed. Patients were divided into two groups according to the Centre for Disease Control and Prevention (CDC) criteria for AIDS-related TE: AIDS patients with suspected neurotoxoplasmosis and AIDS and HIV-negative patients with other confirmed neurological diseases but no suspicions of TE. Predictive values, diagnostic accuracy, sensitivity and specificity of the PCR B1 method were calculated.ResultsThe results obtained from 190 patients showed that this assay has a good sensitivity and specificity (83.3% and 95.7%, respectively) for the diagnosis of TE in AIDS patients.ConclusionPCR using the B1 gene and B22/B23 set of primers is a single, rapid and reliable method that may be valuable for discrimination between toxoplasmosis and other central nervous system (CNS) diseases.

IgG1,IgG2 and IgE intrathecal synthesis in Angiostrongylus cantonensis meningoencephalitis

INTRODUCTION Angiostrongylus cantonensis meningoencephalitis is an emergent zoonotic disease in the Caribbean basin, characterized by the presence of eosinophils and third stage larva of the helmint. OBJECTIVE To analyze the IgG subclasses and IgE intrathecal synthesis patterns obtained by reibergrams in pediatric patients suffering from eosinophilic meningoencephalitis due to A. cantonensis. PATIENTS AND METHODS 20 pediatric patients with the disease were studied. During the first diagnostic lumbar puncture an eosinophilic pleocytosis was found. Simultaneously a serum sample was taken. Eight days later, a second lumbar and venous puncture was performed. IgA, IgM, IgG, albumin in serum and cerebrospinal fluid samples were quantified by immunodiffusion in addition to a differential cell count in cerebrospinal fluid. IgG subclasses were quantified in 10 patients by immunodiffusion and IgE in four patients by nephelometry. RESULTS During the first diagnostic lumbar puncture, all the cases had a blood-cerebrospinal fluid barrier dysfunction with absence of immunoglobulins intrathecal synthesis, a mean of 450 cells/mul and an average of 48% of eosinophils. In the second lumbar puncture 40% of the patients had a dysfunction of the blood-cerebrospinal fluid barrier and an intrathecal synthesis pattern of IgA+IgM+IgG in 50% of the patients. Eight patients had an intrathecal IgA+IgG class response. The synthesis pattern of IgG subclasses was IgG1+IgG2 in six patients, IgG1+IgG2+IgG3 in one patient, IgG1+IgG2+IgG4 in one more patient. Two patients from the second lumbar puncture remained without intrathecal synthesis. IgE intrathecal synthesis was observed in the four analyzed patients in the first diagnostic lumbar puncture. CONCLUSIONS The IgG1+IgG2 and IgE intrathecal synthesis pattern demonstrated the complexity of the antigenic mosaic of the helmint and it can contribute to diagnosis of eosinophilic meningoencephalitis due to A. cantonensis.

C3c intrathecal synthesis evaluation in patients with multiple sclerosis

INTRODUCTION Multiple sclerosis (MS) is a chronic, inflammatory and progressive disease of the central nervous system in which local inflammatory injuries of the brain white matter appears, being the most outstanding feature the myeline loss (demyelination). OBJECTIVE To determine if the complement system might be involved in the MS immunopathogeny favouring the mechanism intervening in the myelin destruction. METHOD Samples of sera and CSF from twelve patients with a diagnosis of MS obtained at the moment of the admission to the hospital at the beginning of the break out, were collected. Levels of C3c and albumin in sera and in CSF were quantified using radial immunodiffusion plates. RESULTS High values over 80% of intrathecal synthesis were obtained except in one of the patients. CONCLUSION Intrathecal synthesis of C3c and its liberation to the CSF means that the activation of the complement system in any of the two ways has taken place, and that once performed its biological functions, has suffered a degradation process.

Mannan-binding lectin in cerebrospinal fluid: a leptomeningeal protein

BackgroundMannan-binding lectin (MBL), a protein of the innate immune response is attracting increasing clinical interest, in particularly in relation to its deficiency. Due to its involvement in brain diseases, identifying the source of MBL in CSF is important. Analysis of cerebrospinal fluid (CSF) can provide data that discriminates between blood-, brain-, and leptomeninges-derived proteins. To detect the source of MBL in CSF we need to consider three variables: the molecular size-dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MBL concentration correlation with the albumin CSF/serum quotient (QAlb), i.e., with CSF flow rate.MethodsMBL was assayed in samples of CSF and serum with an ELISA, coated with anti MBL antibodies. Routine parameters such as albumin-, immunoglobulin- CSF/serum quotients, oligoclonal IgG and cell count were used to characterize the patient groups. Groups comprised firstly, control patients without organic brain disease with normal CSF and normal barrier function and secondly, patients without inflammatory diseases but with increased QAlb, i.e. with a blood CSF barrier dysfunction.ResultsMBL concentration in CSF was at least five-fold higher than expected for a molecular-size-dependent passage from blood. Secondly, in a QIgM/QAlb quotient diagram (Reibergram) 9/13 cases showed an intrathecal fraction in some cases over 80% of total CSF MBL concentration 3) The smaller inter-individual variation of MBL concentrations in CSF of the control group (CV = 66%) compared to the MBL concentrations in serum (CV = 146%) indicate an independent source of MBL in CSF. 4) The absolute MBL concentration in CSF increases with increasing QAlb. Among brain-derived proteins in CSF only the leptomeningeal proteins showed a (linear) increase with decreasing CSF flow rate, neuronal and glial proteins are invariant to changes of QAlb.ConclusionsMBL in CSF is predominantly brain-derived and all results pointed to the leptomeningeal cells as the source of the protein. The evaluation of this protein requires the interpretation of its absolute concentrations in CSF as a function of the albumin quotient, QAlb. This recognition of MBL in brain cells opens a new field of discussion about the function of the innate immune response in CNS in cases of acute and chronic neurological diseases.

Intrathecal synthesis of IgE in children with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis

BackgroundEosinophilic meningoencephalitis caused by the helminth Angiostrongylus cantonensis, is an emerging infectious disease in America. The objective of this paper was to determine if the intrathecal synthesis of immunoglobulin E is produced during the acute phase of the disease.MethodsThirteen patients, mean age 4.5 years were studied; a diagnostic lumbar puncture was performed and serum samples taken. Immunoglobulin E (IgE) in serum and in cerebrospinal fluid (CSF) was quantified by nephelometry. Control patients had other infections or other neurological diseases.ResultsThe mean cell count in the CSF was 500 × 10-6 cells/L and of these 23% were eosinophils. In blood the eosinophils were 13%. The chief symptoms of the patients were migraine, vomiting and fever and 50% presented some meningeal signs. IgE intrathecal synthesis analyzed by the corresponding quotient diagram (Reibergram) was observed in all patients. No intrathecal IgE synthesis was seen in control patients.ConclusionIntrathecal synthesis of IgE demonstrates the participation of this immunoglobulin in the destruction of the third stage larvae of the parasite in the CSF. The test should be considered in our environment as a tool to aid diagnosis.

CSF/serum quotient graphs for the evaluation of intrathecal C 4 synthesis

BackgroundCerebrospinal fluid (CSF)/serum quotient graphs have been used previously to determine local synthesis in brain of immunoglobulins and C3 complement component. The aim of this study was to use the same technique to construct quotient graphs, or Reibergrams, for the beta globulin C4 and to evaluate the method for assessing intrathecal synthesis in neurological disease.MethodsThe constants in the previously-defined Reibergram for immunoglobulin IgA were used to calculate the CSF/serum quotient for C4. CSF and serum were analyzed for C4, IgA and albumin from a total of 12 patients with meningoencephalitis caused by encapsulated microorganisms and 10 subjects without infections or inflammatory neurological disease, some of which had dysfunction of the blood-CSF barrier,ResultsThe formula and C4 Reibergram with the constants previously found for IgA, determined the intrathecal C4 synthesis in CSF. The intrathecal C4 fraction in CSF (C4 loc in mg/l) was compared to the C4-Index (fraction of CSF: serum for C 4/fraction of CSF: serum for albumin). There was a significant correlation between the two formulae. The CSF/Serum quotient graph was superior for detecting intrathecal synthesis of C4 under variable conditions of blood-CSF barrier permeability.ConclusionThe C4 Reibergram can be used to quantify the intrathecal synthesis of this component of the complement system in different infectious diseases of the central nervous system and is especially useful for patients with blood-brain barrier dysfunction.

Reibergrama para la evaluación de la síntesis intratecal de C3c

RESUMEN - I n t roduccion: El diagrama de las razones de Reiber o re i b e rgrama cobra cada dia mayores usospara la caracterizacion de la sintesis intratecal de proteinas. El re i b e rgrama fue definido para las clasesm a y o res de inmunoglobulinas pero luego ha sido utilizado para evaluar otras proteinas basado en la teoriade la difusion molecular/velocidad de flujo del liquido cefalorraquideo (LCR). Metodo: El C3c, productode la degradacion del factor del complemento C3 y con una masa molecular de 145 KDa, se acerca a lascaracteristicas moleculares de la IgG para las leyes de la difusion de Fick. Se asume las constantes de la IgGen la formula de Reiber para evaluar la sintesis intratecal de C3c asi como su correspondiente re i b e rg r a m a .Se estudiaron 27 pacientes y 27 controles a los que se les dosifico albumina y C3c en suero y LCR porinmunodifusion radial. Resultados: Con el re i b e rgrama propuesto para el C3c se evaluaron estos pacientes.Se comprueba la validez de este re i b e rgrama para distintas condiciones de barrera con o sin sintesis intratecalde C3c.

Angiostrongyliasis in the Americas

To the Editor: We read with special interest the article by Hochberg et al. about angiostrongyliasis in Hawaii (1). Angiostrongylus cantonensis meningitis in the Americas was reported by Aguiar et al. in Cuba in 1981 (2), and we have studied this zoonosis during the ensuing 25 years. We agree with the authors about the difficulty in obtaining a specific immunoassay for detection of antibodies to A. cantonensis antigens. In Cuba, as in Hawaii, no other cause of eosinophilic meningitis was identified.

Comparison of Major Immunoglobulins Intrathecal Synthesis Patterns in Ecuadorian and Cuban Patients with Angiostrongyliasis

Angiostrongylus cantonensis meningitis was first reported in Cuba in 1981, and it was recently reported in South America. The aim of this paper is to evaluate the intrathecal immunoglobulin synthesis patterns from Cubas and Ecuadors patients with angiostrongyliasis; 8 Ecuadorian patients from two different outbreaks and 28 Cuban patients were studied. Simultaneous blood and cerebrospinal fluid samples were taken. Immunoglobulin (Ig) A, IgM, IgG, and albumin were quantified by radial immunodiffusion. Corresponding Reibergrams were applied. A three-Ig pattern was the most frequent in the two groups, but IgM was presented in all Ecuadorian young mature patients; however, in the Cuban children, only 12 of 28 patients had intrathecal IgM, but about 90% had an IgA and IgG synthesis at time of later puncture. This indicates that, with a larger amount of parasites ingested, clinical symptoms are more severe, and a higher frequency of intrathecal IgM synthesis could be observed. This is discussed as a similarity with the intrathecal IgM synthesis in African trypanosomiasis.

Intrathecal Activation as a Typical Immune Response within the Central Nervous System in Angiostrongyliasis

Angiostrongylus cantonensis is a zoonotic pathogen that occasionally causes human angiostrongyliasis; its main clinical manifestation is eosinophilic meningitis. This report defines the concept of intrathecal activation of complement as evidence of intrathecal synthesis of major immunoglobulins during this disease. Details are presented of the activation of complement system components in cerebrospinal fluid, and their application to our understanding of this tropical disease, which is emerging in the Western hemisphere. Intrathecal synthesis of at least one of the major immunoglobulins and a wide spectrum of patterns may be observed. Although intrathecal synthesis of C3c is always present, C4 intrathecal synthesis does not occur in every patient. The diversity of intrathecal synthesis and activation of the different complement pathways enables their division into three variant groups (A, B, and C). Variant group A includes the classical and/or lectin pathway and involves two or more major immunoglobulins with C3 and C4 intrathecal synthesis. Variant group B involves C4 in cerebrospinal fluid that comes from blood in the intrathecal activation of the classical pathway. Variant group C includes the alternative pathway.