Detección de anticuerpos

Un número limitado de enfermedades (infección fúngica y alergia), que se señalan a continuación, se benefician de la detección de anticuerpos:

• El diagnóstico y la monitorización del tratamiento de la coccidioidomicosis;
• El diagnóstico y la monitorización del tratamiento de la aspergilosis pulmonar crónica;
• Diagnóstico y manejo de la aspergilosis broncopulmonar alérgica;
• El diagnóstico y la monitorización del tratamiento de la rinosinusitis alérgica, crónica granulomatosa causada por Aspergillus;
• Información complementaria para el diagnóstico de las bronquitis por Aspergillus en pacientes inmunocompetentes;
• Diagnóstico de la histoplasmosis aguda y crónica;
• Diagnóstico de la paracoccidioidomicosis.

Existen otras pruebas de detección de anticuerpos, pero tienen una utilidad muy limitada en la práctica clínica. Algunas, como la detección de anticuerpos frente a Pneumocystis o Cryptococcus han sido útiles en estudios que han analizando la exposición previa, especialmente en niños. Algunas se han combinado con la detección de antígenos para hacer kits diagnósticos como la de anticuerpos anti manano de Candida. Otras se han usado para diagnosticar infecciones raras, como la detección de anticuerpos en LCR en la meningitis por Sporothrix, la detección de anticuerpos frente a Scedosporium para diagnosticar la bola fúngica causada por S. apiospermum o para distinguir los diferentes hongos que causan el micetoma.

Visualiza nuestro video mostrando la técnica de inmunodifusión (amablemente realizado por Oscar Zaragoza, del Laboratorito de Referencia del Instituto de Salud Carlos III de Madrid, España, ozaragoza@isciii.es).

Formatos de las técnicas

Se han utilizado diversos formatos para la detección de anticuerpos en suero, incluyendo la inmunodifusión doble (DD) o la inmunodifusión (ID), la contrainmunoelectroforésis (CIE), el enzima inmunoensayo (ELISA) y la fijación de complemento (CF). Con menor frecuencia, los métodos usados han sido la hemaglutinación, el radioinmunoensayo, el inmunobloting y la co-CIE. Las diferentes ventajas e inconvenientes, así como las fortalezas y debilidades de estos métodos se pueden consultar en numerosos libros de texto y no van a ser discutidas aquí.

Tipos de inmunoglobulina

La mayoría de las pruebas detectan IgG o IgE, con la excepción de la IgM para la coccidioidomicosis. No se ha encontrado ninguna utilidad en la detección de IgA frente a los hongos. Es posible que la detección de IgM tenga más utilidad clínica que la conseguida actualmente, especialmente para la aspergilosis, pero no esta disponible de forma rutinaria.

Detección de anticuerpos frente a Coccidioides

La detección de anticuerpos frente a Coccidioides es clave en el diagnóstico de la enfermedad. Se pueden detectar anticuerpos en fases iniciales de la clase IgM y tardíos de la clase IgG. En la coccidioidomicosis primaria, la IgM se suele detectar en aproximadamente el 50% de los casos en la primera semana y en el 90% de los casos en la tercera semana, tras la aparición de los síntomas, excepto si el paciente está inmunocomprometido. La IgG se puede detectar entre la segunda y la vigésimo octava semana tras el comienzo de la enfermedad. Los resultados negativos no sirven para descartar la enfermedad, especialmente en la fase aguda. La sensibilidad global de la serología de la coccidioidomicosis es aproximadamente de un 82%. La detección de anticuerpos mediante ELISA es, probablemente, la técnica más sensible pero menos específica si se compara con la detección de precipitinas en tubo que detecta IgM, y la CF que detecta IgG.

Una de las ensayos en el mercado se hace por Meridian.

La primera y mas específica característica de la meningitis es la detección de IgM en el LCR. Hay que pensar específicamente en esta enfermedad para no errar el diagnóstico ya que el examen directo y el cultivo suelen ser negativos, y el recuento celular, la concentración de proteínas y glucosa son consistentes con la meningitis tuberculosa.

Monitorización de la respuesta al tratamiento

La titulación seriada de los anticuerpos mediante CF es útil en la evaluación de la evolución de la infección y de la eficacia del tratamiento antifúngico. El análisis en paralelo de muestra previas y nuevas es importante para asegurar la consistencia de los resultados. El aumento de los títulos en la CF sugiere empeoramiento o diseminación, mientras que la disminución de los mismos suele indicar respuesta terapéutica.

Detección de anticuerpos frente a Aspergillus

Hay muchas pruebas comercializadas de detección de anticuerpos IgG frente a A. fumigatus. Hay disponibles un número pequeño de pruebas, que no están validadas completamente, para otras especies de Aspergillus incluyendo A. flavus, A. terreus, A. niger, A. versicolor y A. clavatus. Todas las técnicas se realizan en el suero del paciente.

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Factsheets

Coccidioides

OVERVIEW Antibody detection is key to the diagnosis of several manifestations of coccidioidomycosis. Early (IgM) and late (IgG) antibodies may be produced. – IgM becomes detectable usually between the first (~50%) and third (~90%) weeks after symptoms in primary coccidioidal infection, except in the immunocompromised patient. – IgG becomes detectable between the second and 28th week post onset of illness.

AVAILABLE ASSAYS – EIA: Meridian; MVista Coccidioides Antibody IgG IgM EIA – POCT: IMMY sona™ rapid (<30 mins) LFD for both IgG and IgM (sensitivity ~100%; specificity ~77%)

DIAGNOSIS – Coccidioidal meningitis: the earliest specific feature is IgM antibody in CSF. As microscopy and culture are usually negative, and the cell count, protein and glucose are consistent with tuberculous meningitis, the diagnosis can be missed.

MONITORING TREATMENT Serial titration of CF antibody is useful in the evaluation of improvement of worsening of disease, and in evaluation of efficacy of antifungal therapy. Parallel testing of previously collected and new specimens may be required to ensure consistency. Increasing CF titers suggest worsening or dissemination; decreasing titers indicate therapeutic response.

PERFORMANCE & INTERPRETATION Negative serologic results cannot be used to rule out coccidioidomycosis (particularly early in the acute phase). The overall sensitivity of anticoccidioidal serologic studies is ~82%. ELISAs for detection of both IgM and IgG are probably the most sensitive of the methods, compared with tube precipitins for IgM and CF for IgG, but may be less specific.

Aspergillus

OVERVIEW IgG testing of serum is a mainstay in the diagnosis of chronic pulmonary aspergillosis

AVAILABLE ASSAYS – A. fumigatus IgG: Immy, Serion/Virion, Bioenche, BioRad, Thermofisher, Elitech, Microgen, Meridian, Siemens, NovaTec and Bordier.  A commercial western blot assay is also available: LDBio

DIAGNOSIS SPECIES: There are multiple marketed IgG antibody tests to detect A. fumigatus antibodies. A smaller number of less well used and often usually incompletely validated tests are available for other species of Aspergillus including A. flavus, A. terreus, A. niger, A. versicolor and A. clavatus. SAMPLE: All testing is on serum. DETECTING SENSITISATION: IgE antibody testing against A. fumigatus is useful to detect Aspergillus sensitisation. The skin prick test against A. fumigatus is more sensitive than blood testing. Either is required for the diagnosis of ABPA (usually both are positive) and are usually positive at a much lower level in patients with severe asthma with fungal sensitisation (SAFS). This test could be a useful screening test in asthmatic patients to detect ABPA or SAFS, if skin testing not done (IgE measurement) CPA: Some patients with other Aspergillus diseases, notably chronic pulmonary aspergillosis, have positive Aspergillus IgE antibody titres. Occasionally this test is positive when the IgG antibody test is negative, which is helpful (view Skin testing and IgE testing)

MONITORING TREATMENT Falling IgG antibody titre is useful as a measure of therapeutic response in chronic pulmonary aspergillosis and Aspergillus rhinosinusitis. The rate of fall is slow and takes weeks or months even in patients doing well. Lack of fall is suspicious of treatment failure.

PERFORMANCE & INTERPRETATION The best IgG assays have a 90-95% sensitivity for chronic pulmonary aspergillosis and aspergilloma caused by A. fumigatus, much more sensitive than culture. Requires compatible radiology and relatively non-immunocompromised patient for the diagnosis to be made. The antibody titre varies widely.  30-50% of patients with ABPA have detectable A. fumigatus antibodies, usually at low titre. High titres suggest the complication of chronic pulmonary aspergillosis, which needs radiological correlation.  Fungal sinusitis is more often caused by A. flavus than A. fumigatus. A. flavus IgG antibody is useful confirmatory evidence of infection, especially if cultures are negative but histology or microscopy positive.  Patients with Aspergillus bronchitis often have positive IgG antibodies (and negative IgE antibodies) but A. fumigatus is slightly less common as the cause, so non-fumigatus IgG antibodies (precipitins) may be required.  The optimum cut-off for positive IgG titres varies and may be higher than some manufacturers recommend, because healthy control sera (which almost always have very low antibody titres) may have a low titre in which is too low in specific groups. An example is cystic fibrosis in which the baseline Aspergillus IgG antibody titres are higher than normal controls.

Candida

OVERVIEW Antibodies to Candida species, both agglutinins and precipitins, have been found in patients with and without systemic candidiasis. There are biochemical differences between the mycelial and blastospore forms of Candida albicans (Chattaway et al., 1968). The antigenicity of the two growth phases of Candida albicans differ in qualitative and quantitative ways with a specific mycelial antigenic component having diagnostic potential (Evans et al., 1973). Specific mycelial antigens are superior to yeast cell antigens in the diagnosis of suspected systemic candidosis (Umenai & Chiba 1977; Syverson, Buckley & Campbell 1975). These and later data (Peman et al., 2011) lead to the commercialisation of a Candida albicans indirect chemiluminescent immunoassay for the detection of antibodies against antigens located on the cell wall surface of the mycelium of Candida albicans (CAGTA [Candida albicans germ tube antibody]) in human serum or plasma (Vircell, Spain).

AVAILABLE ASSAYS GaDia CandiDia Platelia™ Candida Ab Era Biology Candida Ab Dynamiker Candida Ab

DIAGNOSIS

MONITORING TREATMENT

PERFORMANCE & INTERPRETATION The test appears to discriminate between infection and colonization and it has a high negative predictive value (73%). Combining the CAGTA assay with the  ß-1-3-D-glucan test (Fungitell, Associates of Cape Cod, USA) in patients on empirical antifungal therapy for presumed candidiasis has shown that serial determination of CAGTA and glucan during empirical antifungal therapy has a high sensitivity (93%) and negative predictive value (91-97%). If properly confirmed, this strategy could be used to discontinue antifungal treatment in at least 30% of patients as a complementary tool in antifungal stewardship programmes (Martinez-Jiménez et al., 2015; supplementary data).

Histoplasma

OVERVIEW Antibody detection is useful for the chronic forms of histoplasmosis, notably chronic pulmonary histoplasmosis and some patients with (subacute) progressive disseminated histoplasmosis. Seroconversion is often documented after acute pulmonary histoplasmosis.

AVAILABLE ASSAYS Antibody kits: Meridien;  Immy; Miravista

DIAGNOSIS Seroconversion from negative to positive is the most straightforward means of making a diagnosis of acute pulmonary histoplasmosis but it may take 2 to 6 weeks to seroconvert. Over 90% of patients with chronic pulmonary histoplasmosis have detectable antibodies to Histoplasma. About 30-70% of patients with disseminated histoplasmosis have detectable antibodies, depending on the immunological status of the host.

PERFORMANCE & INTERPRETATION Cross reaction is common with sera from patients with blastomycosis, penicilliosis and paracoccidioidomycosis and so the test cannot be used to differentiate these infections. Both complement fixation (CF) and immunodiffusion (ID) antibodies may be positive, without a clear advantage between them in sensitivity, but specificity appears to be higher with ID. If one test is negative the other may be positive.

Paracoccidioides

OVERVIEW Several serological assays detecting antibody have been used for the early diagnosis of Paracoccidioides brasiliensis infection. Standardisation has been difficult because of variations in assay sensitivity, specificity and reproducibility mostly related to lot to lot variations in the preparation of antigens required for the assays. The most frequently used test is double immunodiffusion assay which has a sensitivity and specificity ranging from ~80 to 90%. Antigens preparations containing high concentration of the 43 KDa glycoprotein are required to optimize performance of the test.

AVAILABLE ASSAYS

DIAGNOSIS

MONITORING TREATMENT Falling IgG antibody titres is useful as a measure of therapeutic response in acute and chronic forms of paracoccidioidomycosis. The rate of fall is slow and takes months or years even in patients doing well. Lack of fall in antibody titre is suspicious of treatment failure.

PERFORMANCE & INTERPRETATION ELISA has a higher sensitivity but false-positive results have been frequently reported in normal individuals living in endemic areas for P. brasiliensis as well as in patients with tuberculosis, leprosy, leishmaniasis and other medical conditions. In home made tests, the following results were obtained with 3 different test formats View paper  These data are consistent with a useful role for antibody testing in acute and chronic forms of paracoccidioidomycosis in which sensitivity of different assays may exceed 90% . Despite the good results of serology obtained with patients co-infected with paracoccidioidomycosis and AIDS in this publication, other experienced laboratories describe limited sensitivity of antibody detection in the presence of comorbidities associated with immunodeficiency.