Accuracy of serological tests for diagnosis of chronic pulmonary aspergillosis: A systematic review and meta-analysis.

Chronic pulmonary aspergillosis (CPA) is a slow and progressive disease that develops in preexisting lung cavities of patients with tuberculosis sequelae, and it is associated with a high mortality rate. Serological tests such as double agar gel immunodiffusion test (DID) or counterimmunoelectrophoresis (CIE) test have been routinely used for CPA diagnosis in the absence of positive cultures. However, these tests have been replaced with enzyme-linked immunoassay (ELISA) and, a variety of methods. This systematic review compares ELISA accuracy to reference test (DID and/or CIE) accuracy in CPA diagnosis. It was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The study was registered in PROSPERO under the registration number CRD42016046057. We searched the electronic databases MEDLINE (PubMed), EMBASE (Elsevier), LILACS (VHL), Cochrane library, and ISI Web of Science. Gray literature was researched using Google Scholar and conference abstracts. We included articles with patients or serum samples from patients with CPA who underwent two serological tests: ELISA (index test) and IDD and/or CIE (reference test). We used the test accuracy as a result. Original articles were considered without a restriction of date or language. The pooled sensitivity, specificity, and summary receiver operating characteristic curves were estimated. We included 14 studies in the review, but only four were included in the meta-analysis. The pooled sensitivities and specificities were 0.93 and 0.97 for the ELISA test. These values were 0.64 and 0.99 for the reference test (DID and/or CIE). Analyses of summary receiver operating characteristic curves yielded 0.99 for ELISA and 0.99 for the reference test (DID and/or CIE). Our meta-analysis suggests that the diagnostic accuracy of ELISA is greater than the reference tests (DID and/or CIE) for early CPA detection.

Evaluation of a multiplex flow immunoassay versus conventional assays in detecting autoantibodies in systemic lupus erythematosus.

INTRODUCTION: Conventional diagnostic assays are being replaced with automated multiplex assays, but their performance needs to be evaluated. We compared a multiplex flow immunoassay with conventional techniques in the detection of antinuclear antibodies (ANAs) and antibodies to specific extractable nuclear antigens (ENAs) in serum samples from patients with systemic lupus erythematosus. METHODS: A total of 140 consecutive Chinese patients with systemic lupus erythematosus and 41 healthy controls were included. The automated BioPlex 2200 ANA Screen assay (Bio-Rad Laboratories, Hercules [CA], US) was compared with indirect immunofluorescence. In addition, use of BioPlex 2200 to detect anti-ENA antibodies was compared with in-house assays of countercurrent immunoelectrophoresis (CIEP), enzyme-linked immunosorbent assay (ELISA), and line blot. RESULTS: The sensitivity and specificity of BioPlex in detecting ANAs (91.4% and 95.1%, respectively) were comparable to those of indirect immunofluorescence (90.7% and 85.4%, respectively). Overall, BioPlex achieved the best agreement with ELISA in detecting anti-ENA antibodies: agreement was >90% for most antibody types (κ=0.79-0.94). In contrast, agreement was poorest with CIEP, ranging from 85.6% (κ=0.33) for anti-Sm antibodies to 93.9% (κ=0.88) for anti-Ro antibodies. Overall, BioPlex and ELISA had the highest sensitivity, whereas CIEP had the highest specificity. In terms of disease association, anti-Sm detected by CIEP had the best positive predictive value and specificity for lupus nephritis. CONCLUSIONS: In a local lupus cohort, BioPlex showed comparable sensitivity to indirect immunofluorescence in detecting ANAs and comparable performance to ELISA in detecting anti-ENA antibodies. However, CIEP was the best method in terms of disease specificity.

The role of latex agglutination test for the etiological diagnosis of pleural effusion in children and adolescents.

INTRODUCTION: The etiological diagnosis of pleural effusion is a difficult task because the diagnostic tools can only establish a definitive etiological diagnosis in at most 76% of cases. OBJECTIVES: To verify the diagnostic accuracy of the latex agglutination test (LAT) for the etiological diagnosis of pleural effusions caused by Streptococcus pneumoniae and Haemophilus influenzae type b. METHODS: After thoracocentesis, paired fresh samples of pleural fluid from 418 children and adolescents were included in this investigation. They were tested blindly and simultaneously through counterimmunoelectrophoresis (CIE) and LAT for both bacteria. Sensitivity, specificity, predictive values and likelihood ratios (LR) were calculated taking CIE as a reference standard. RESULTS: The sensitivity and specificity of LAT was 100% (95% confidence interval, 94.4%-100%) and 83.3% (95% confidence interval, 79.0%-87.0%), respectively, whereas the positive (calculated from Bayes' theorem) and negative predictive values were, respectively, lower than 1% and 100% (95% confidence interval, 98.8%-100%). Positive and negative LR were 6.0 (95% confidence interval, 4.7-7.6) and zero, respectively. CONCLUSIONS: Our results suggest that LAT is a useful tool for the etiological diagnosis of pleural effusion. It is a reliable, rapid, simple to perform and shows an excellent yield in our studied population, helping to prescribe appropriate antibiotics for this clinical condition.

[Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium]. / Diagnóstico serológico de la esporotricosis mediante el empleo del antígeno de micelio de Sporothrix schenckii sensu stricto.

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

Diagnóstico serológico de la esporotricosis mediante el empleo del antígeno de micelio de Sporothrix schenckii sensu stricto / Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium

En este estudio se desarrolló y se evaluó el ensayo por inmunoabsorción ligado a enzimas (ELISA), para la detección de anticuerpos en sueros de pacientes con esporotricosis, para lo cual se empleó un antígeno crudo de Sporothrix schenckii sensu stricto obtenido a partir de la forma micelial. Los sueros positivos para esporotricosis fueron ensayados por otras técnicas serológicas: inmunodifusión doble (IDD) y contrainmunoelectroforesis (CIE). El ensayo fue validado utilizando sueros de otras patologías como histoplasmosis, paracoccidioidomicosis, tuberculosis, leishmaniasis, lupus y sueros de individuos sanos como controles negativos. Se encontró una especificidad de 100 % con las técnicas utilizadas y una sensibilidad del antígeno de S.schenckii sensu stricto, por encima del 98% para IDD, CIE y ELISA. Estos resultados demuestran la alta sensibilidad y especificidad del antígeno de S. schenckii sensu stricto, para el diagnóstico de la esporotricosis, empleando las técnicas de IDD, CIE y ELISA. Los resultados sugieren, que este antígeno podría ser usado en conjunto con otras pruebas convencionales para el diagnóstico diferencial y puede ser útil para monitorizar la evolución de la enfermedad y respuesta al tratamiento.

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

A comparative study on microscopic agglutination test and counterimmunoelectrop- horesis for early detection of human leptospirosis.

BACKGROUND AND OBJECTIVES: Leptospirosis is a potentially fatal bacterial disease that mimics many diseases; therefore, laboratory confirmation is pivotal. Though microscopic agglutination test (MAT) is accepted as World Health Organisation (WHO) reference test, it has got many pitfalls such as being hazardous, tedious, cumbersome and expensive. Counterimmunoelectrophoresis (CIE) is popularly used for diagnosing many infectious diseases but rarely for Leptospirosis. The aim of this study is to find suitability of CIE for the routine laboratory diagnostic purposes. MATERIALS AND METHODS: Repeat sampling (paired sera) was possible from 401 subjects of which 181 were in-patients of Salem Government General and Private Hospitals and the remaining 220 MAT negative healthy College students gave their consent for the study. All the 802 sera samples were collected from January 2009 to November 2012 and subjected to the present study. After carrying out MAT and CIE on the suspected and control samples, a comparative evaluation was conducted. McNemars test method was used to find out the significant difference between the two tests in the early diagnosis. RESULT: The sensitivity, specificity, Positive Predictive value (PPV), Negative Predictive value (NPV) and Efficiency test for CIE were 96.80%, 89.28%, 95.23%, 92.59% and 94.47%, respectively. The corresponding values for MAT were 95.90%, 89.83%, 95.08%, 91.37% and 93.92%, respectively. There was no significant difference between MAT and CIE at 95% and 99% confidence intervals according to McNemars test. P value in the early stage of illness was greater for CIE than MAT when Polymerase Chain Reaction (PCR) was used as Gold Standard of diagnosis. INTERPRETATION AND CONCLUSION: It was concluded that the CIE could be advantageous over MAT due to its safety, rapidity, simplicity, economic and easy for large number of samples. It can answer little earlier than MAT and found as reliable as that of MAT. Since both the tests had shown similar efficacies in the later stage of the illness, the importance could be given to CIE due to early diagnosis.

Performance of a multiplex assay compared to enzyme and precipitation methods for anti-ENA testing in systemic lupus and systemic sclerosis.

INTRODUCTION: Testing for autoantibodies to extractable nuclear antigens (ENA) is essential in the investigation of connective tissue disease. Counterimmunoelectrophoresis is an early described testing methodology for antibodies to ENAs, but is labour-intensive, only moderately sensitive, and reliant on high-quality reference sera. Enzyme-linked immunosorbent assay (ELISA) is automatable for relatively high sample throughput, but has issues with false positives. The addressable laser bead immunoassay (ALBIA) is a multiplex technology which can assess several antibody specificities simultaneously on a small serum sample. We report performance of an ALBIA system compared with CIE and ELISA. METHODS: Samples from 100 systemic sclerosis patients attending Royal Free Hospital in 2007 and 99 SLE patients attending St Thomas's Hospital in 2007-2008 were studied. All samples were tested for antibodies to RNP, Sm, Ro, La, Scl-70, Jo-1 by in-house CIE, FIDIS™ ALBIA (BMD, France), and ELISAs (Phadia, Germany). Cohen's kappa coefficient was used to examine agreement of the different assay methods for the same antibody. McNemar's test was used to detect differences between methodologies. RESULTS: One sample was positive for anti-Jo-1 by CIE, & confirmed by ALBIA & ELISA. All 198 remaining samples were anti-Jo-1 negative by all 3 methods. With respect to RNP, Ro, La, Scl-70 antibodies, there was good agreement in assay performance between CIE, ALBIA, and ELISA. For Sm, agreement was less good between CIE and ELISA (kappa 0.491), and ALBIA and ELISA (kappa 0.403). Using McNemar's test performance was no different between the 3 assays, with the following exceptions: between CIE and ELISA for Ro-60 (p<0.01) and RNP (p<0.05), and between ALBIA and ELISA for RNP (p<0.01). CONCLUSIONS: The FIDIS™ ALBIA produced similar level of performance as CIE, but with advantages of automation, and less dependence on highly skilled operators. ALBIA represents a potential advancement applicable to routine Immunology diagnostics.

Assessment of four serological techniques in the immunological diagnosis of farmers' lung disease.

Farmers' lung disease (FLD) is a pulmonary disease that results from repeated inhalation of antigens from mouldy hay or straw. The objective of this prospective study was to assess the reliability of four serological techniques in FLD diagnosis. Sera from 15 consecutive patients with FLD, 15 healthy control farmers and 30 urban controls were analysed using four serological techniques [electrosyneresis (ES), Ouchterlony double diffusion (DD), ELISA and Western blot (WB)] with four antigens (Absidia corymbifera, Eurotium amstelodami, Wallemia sebi and Saccharopolyspora rectivirgula). In the authors' region, ES on cellulose acetate with A. corymbifera antigen was the most relevant diagnostic tool for discriminating FLD patients from healthy exposed farmers (sensitivity 87 %, specificity 100 %). DD tests were in accordance with ES, but their discriminatory power was lower. No threshold indicating both good sensitivity and specificity could be established with ELISA. WB analysis failed to identify specific bands for FLD. This study demonstrates the efficacy of determining precipitin levels with an appropriate technique, using a panel of antigens consistent with the specific exposure of a given area.

Observation and identification of lactate dehydrogenase anomaly in a postburn patient.

OBJECTIVE: Lactate dehydrogenase (LDH) anomaly is one of the macroenzymes. Macroenzymes are enzymes in serum that have formed high molecular mass complexes, either by self polymerisation or by association with other serum components. The aim of this study was to identify the properties of LDH anomaly and observe the changes from admission to discharge in a postburn patient with LDH anomaly in his serum. METHODS: LDH isoenzymes of the serum were electrophoretically fractionated with terylene cellulose acetate supporting media; LDH anomaly was identified by counter immunoelectrophoresis. RESULTS: An abnormal LDH-4 band and an extra band on the cathode of LDH-5 were observed in the serum of this patient and were found to be part of an LDH-IgG complex. As his symptoms improved, the patient's LDH anomaly gradually disappeared. The appearance and disappearance of the anomaly seemed to be related to the progression of the patient's burns. CONCLUSION: In clinical practice, it is important to keep in mind the possibility of an LDH anomaly in patients when the LDH level is abnormally high or does not seem to be related to the clinical state. Early discovery of an LDH anomaly in a patient's serum may be useful for diagnosis and treatment.

Anti-Ku antibodies in connective tissue diseases: clinical and serological evaluation of 14 patients.

OBJECTIVE: To assess the clinical and serological associations of anti-Ku antibodies. METHODS: Fourteen patients with anti-Ku antibody detected by counterimmunoelectrophoresis (CIE) and immunoblot (IB) were retrospectively evaluated. RESULTS: Patients (13 women, one man) had a mean age of 60.3 years (range 19-83). Seven patients had overlap syndromes: 5 polymyositis/scleroderma (PM/SSc), one systemic lupus erythematosus (SLE)/SSc/PM, and one SLE/PM. Three additional patients had undifferentiated connective tissue disease, 2 primary Sjögren's syndrome (SS), one psoriatic arthritis, and one SSc. The clinical manifestations most frequently recorded were arthralgias (86%), myositis (50%) and Raynaud's phenomenon (78.6%). Five patients had esophageal dysmotility, while 6 showed interstitial pulmonary fibrosis (4 of them with reduced DLCO). No case of pulmonary hypertension was observed. All patients had very high titer of ANA with speckled and nucleolar pattern. All the sera were positive for anti-Ku antibodies by CIE: all but one were confirmed by IB. Eight sera contained isolated antibodies to Ku proteins: both subunits were recognized in 7 cases, while isolate reactivity to the 70 kDa protein was detected in one case. Five sera contained additional antibody specificities: anti-Ro 60 kDa in 4 cases, and anti-La/SSB, anti-SL, and anti-PM-Scl in one case each. CONCLUSION: Anti-Ku antibody is found in a wide spectrum of connective tissue diseases including overlap syndromes with SSc and myositis. Raynaud's phenomenon and muscular and joint involvement are the most frequent clinical features associated with anti-Ku antibodies, which are frequently detected in association with anti-Ro/SSA and/or other antinuclear specificities.

Evolución clínica y serológica de la enfermedad mixta del tejido conjuntivo de inicio infantil o juvenil / Clinical and serological course of infantile- or juvenile- onset mixed connective tissue disease

La enfermedad mixta del tejido conjuntivo (EMTC) se caracteriza por la combinación de síntomas de lupus eritematoso sistémico (LES), esclerodermia y polimiositis. Objetivo: Estudiar la evolución clínica y serológica de los pacientes con EMTC de inicio juvenil. Métodos: De los 57 pacientes diagnosticados de EMTC entre los años 1988 y 2001, hemos seleccionado aquellos cuya enfermedad se inició entre los 8 y 18 años de edad. Los anticuerpos anti-Ul-RNP y anti-Sm fueron detectados por contrainmunoelectroforesis (CIE), inmunotransferencia y ELISA (enzimoinmunoánalisis).Resultados: 21 de los 57 pacientes (36,8 por ciento) tuvieron un inicio infantil o juvenil. La edad media de inicio es 12,5 años (8-18) y el tiempo medio de evolución es 13,3 años (1-31). Las principales manifestaciones clínicas fueron artritis (90 por ciento), fenómeno de Raynaud (90 por ciento), miositis (76 por ciento), enfermedad intersticial pulmonar (70 por ciento) y edema en manos (66 por ciento). Todos los pacientes cumplen criterios de LES (76 por ciento), polimiositis (52 por ciento) o esclerodermia (42 por ciento) al final del estudio. Asimismo, todos presentan anticuerpos anti-Ul-RNP por CIE y ELISA (clase IgG). La inmunotransferencia detecta anticuerpos IgG anti-70 kD-Ul-RNP en 15 (71 por ciento), anti-A en 17 (80 por ciento) y anti-C en 9 (42 por ciento). Seis pacientes con criterios de LES tienen anticuerpos anti-D-Sm de clase IgG por inmunotransferencia y por ELISA (clase IgG). Una paciente falleció por vasculitis cerebral. Conclusiones: La evolución clínica sugiere que la EMTC juvenil es un síndrome de solapamiento. La asociación de edema en manos, fenómeno de Raynaud, enfermedad intersticial pulmonar, artritis, miositis y una respuesta anti-Ul-RNP constante á lo largo de la evolución sugieren que la EMTC juvenil es una entidad definida (AU)

Risk of congenital complete heart block in newborns of mothers with anti-Ro/SSA antibodies detected by counterimmunoelectrophoresis: a prospective study of 100 women.

OBJECTIVE: To assess the true prevalence of congenital complete heart block (CCHB) in infants of anti-Ro/SSA-positive women known to have connective tissue disease (CTD) and, secondarily, to evaluate the prevalence of other electrocardiographic abnormalities in these newborns at birth. METHODS: A prospective study was conducted in 4 referral hospitals. One hundred anti-Ro/SSAA-positive mothers were followed up before they became pregnant and during the index pregnancy. Counterimmunoelectrophoresis and immunoblotting were used to test for antibodies to extractable nuclear antigens. RESULTS: Of the 100 women with anti-Ro/SSA antibodies, 2 had infants who developed CCHB in utero (2%). The CCHB was detected at 22 weeks and 20 weeks, respectively. One of the 2 mothers had primary Sjögren's syndrome (SS), and the other had undifferentiated CTD (UCTD). No case of CCHB occurred among the infants of 53 mothers with systemic lupus erythematosus (SLE). No fetal death occurred due to CCHB. In 2 centers, electrocardiography was recorded in 24 unselected newborns, and 4 were found to have sinus bradycardia. CONCLUSION: The prevalence of CCHB in newborns of prospectively followed up women already known to be anti-Ro/SSA positive and with known CTD was 2%. This finding is useful with regard to preconception counseling of these women. The risk of delivering an infant with CCHB may be higher in mothers with primary SS or UCTD than in those with SLE. Additional electrocardiographic abnormalities such as sinus bradycardia and prolongation of the QT interval may be present in their children.

Countercurrent supercritical fluid extraction and fractionation of alcoholic beverages.

A procedure for the recovery of aromatic extracts from distilled alcoholic beverages by means of a countercurrent supercritical fluid extraction (CC-SFE) on a pilot plant scale is studied. The beverage is directly in contact with the carbon dioxide current in a packed column, and the extracts are recovered in two different fractionation cells, where the depressurization occurs. The proposed method allows the selective extraction of aromatic components of the brandy flavor, rendering a high-value concentrated extract and a colored residue without brandy aroma. The content in ethanol of the aromatic extract can be modified by tuning the extraction/fractionation conditions, rendering from 15 to 95% recovery. The effect of the main variables, including extraction pressure and quality of extracting CO(2), has been tested.

Glycosidically bound flavor compounds of cape gooseberry (Physalis peruviana L.).

The bound volatile fraction of cape gooseberry (Physalis peruviana L.) fruit harvested in Colombia has been examined by HRGC and HRGC-MS after enzymatic hydrolysis using a nonselective pectinase (Rohapect D5L). Forty bound volatiles could be identified, with 21 of them being reported for the first time in cape gooseberry. After preparative isolation of the glycosidic precursors on XAD-2 resin, purification by multilayer coil countercurrent chromatography and HPLC of the peracetylated glycosides were carried out. Structure elucidation by NMR, ESI-MS/MS, and optical rotation enabled the identification of (1S,2S)-1-phenylpropane-1,2-diol 2-O-beta-D-glucopyranoside (1) and p-menth-4(8)-ene-1,2-diol 1-O-alpha-L-arabinopyranosyl-(1-6)-beta-D-glucopyranoside (2). Both glycosides have been identified for the first time in nature. They could be considered as immediate precursors of 1-phenylpropane-1,2-diol and p-menth-4(8)-ene-1,2-diol, typical volatiles found in the fruit of cape gooseberry.

Novedoso esquema de inmunización para la obtención de anticuerpos anti-LPS de Haemophilus influenzae tipo B / Novel immunization outline for the obtaining of antibodies anti-LPS of Haemophilus influenzae type B

Se ensayó un novedoso esquema de inmunización dirigido a la elevación de anticuerpos para Haemophilus influenzae tipo b, en el que se combinaron como inmunógenos un antígeno celular inactivado con formaldehído 0,5 por ciento, y un antígeno lipopolisacarídico parcialmente purificado mediante un método de precipitación selectiva con solventes orgánicos. Se obtuvieron anticuerpos de elevada especificidad y actividad biológica con títulos de hasta 1:256 por contrainmunoelectroforesis, que fueron purificados mediante precipitación selectiva con sulfato de amonio y cromatografía de intercambio iónico con la utilización de una columna de DEAE-Sepharose. La metodología propuesta en este trabajo permite la producción de anticuerpos purificados, útiles en la optimización de técnicas de diagnóstico serológicas y de serotipaje de cepas de H. influenzae b(AU)