Evaluation of Serum Galactomannan Enzyme Immunoassay at Two Different Cut-offs for the Diagnosis of Invasive Aspergillosis in Patients with Febrile Neutropenia

Background: Invasive aspergillosis (IA) is an increasingly common and fatal opportunistic fungal infection in patients with haematological diseases. Early diagnosis is difficult as mycological culture techniques have low sensitivity and the radiological tools have low specificity. Galactomannan enzyme immunoassay (GEI) detects galactomannan in the human serum with a reported sensitivity and specificity between 30% and 100%. Aims: The aim of this study was to analyse the role of GEI in diagnosis of IA in patients with febrile neutropenia and to evaluate the role of GEI in the diagnosis of IA as per the revised (2008) European Organization for Research and Treatment of Cancer–Mycoses Study Group (EORTC–MSG) criteria at two different optical density (OD) cut-offs of 0.5 and 1.0. Setting: This prospective study was conducted in Safdarjung Hospital, New Delhi, India. Methods: GEI testing was performed in adult patients of febrile neutropenia with evidence of IA. Results at two different OD indices (ODIs) of 0.5 and 1.0 were analysed. The evaluation of the diagnostic parameter, that is, GEI was measured in terms of sensitivity, specificity and positive and negative predictive value and was validated with the revised (2008) EORTC–MSG diagnostic criteria of IA. Results: One hundred and eleven patients had evidence of IA, of which 79 patients were GEI positive when cut-off ODI was 0.5, whereas with cut-off ODI 1.0, 55 patients were GEI positive. Conclusion: ODI of 1.0 should be considered as positive while in patients with OD between 0.5 and 1.0, repeat sampling from the patient is recommended.     

Diarrhea Associated with Vibrio fluvialis in the United States

We report the isolation in the United States of Vibrio fluvialis from the stools of a patient who had severe watery diarrhea without fever and who subsequently died. V. fluvialis, a known enteric pathogen in other parts of the world, should be suspected in patients with watery diarrhea, especially in coastal areas.     

Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System

The galactomannan enzyme immunoassay (GM-EIA) is widely utilized for the diagnosis of invasive aspergillosis (IA). There is inconsistent reproducibility of results between centers when the assay is processed manually. Automation of EIAs can reduce variation. This study investigated the semiautomation of the GM-EIA on the DS2 (Dynex) platform in the following three stages: (i) DS2 GM-EIA method validation with experimental samples, (ii) DS2 retesting of case-defined clinical samples, and (iii) a 12-month audit of DS2 GM-EIA performance. In stage i, Bland-Altman analysis demonstrated a reduced variance between optical density index (ODI) values for samples processed on two DS2 platforms (mean difference, −0.02; limits of agreement [LOA], −0.19 to 0.14) compared with the variance between samples processed manually and on a DS2 platform (mean difference, 0.02; LOA, −0.25 to 0.3). In stage ii, 100% (14/14 samples) qualitative agreement was observed for serum samples from patients with IA, with no significant change in the ODI values when samples were processed on the DS2 platform. A significant decrease in ODI values was observed for control serum samples on the DS2 platform (difference, 0.01; P = 0.042). In stage iii, a significant reduction in the frequency of equivocal results, from 5.56% (136/2,443 samples) to 1.56% (15/961 samples), was observed after DS2 automation (difference, 4.0%; 95% confidence interval [CI], 2.7 to 5.2%; P < 0.01), with an equivalent increase in negative results. This study demonstrates that GM-EIA automation may reduce intersite variability. Automation does not have an impact on the repeatability of truly positive results but contributes to a reduction in false-positive (equivocal) GM-EIA results, reducing the need to retest a significant proportion of samples.     

Use of a Novel Enzyme Immunoassay Based on Detection of Circulating Antigen in Serum for Diagnosis of Helicobacter pylori Infection

Recently, noninvasive diagnostic tests for Helicobacter pylori infection have gained in significance. We have developed a sensitive and specific noninvasive immunoassay based on the detection of an H. pylori circulating antigen (HpCA) in sera from H. pylori-infected individuals. Monospecific antibody and Western blot analyses were used to demonstrate the presence of the target antigen in H. pylori cell lysate and serum samples. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HpCA in serum. Endoscopic biopsy specimens from the gastric antra of 221 individuals (143 males and 78 females) with dyspeptic symptoms were evaluated for H. pylori infection, with culture used as a “gold standard” for diagnosis. The target H. pylori antigen was identified at 58 kDa. HpCA has been detected by ELISA with high degrees of sensitivity, specificity, and efficiency (>90%), and ELISA results show no significant difference (P > 0.05) from results of H. pylori culture of gastric biopsy specimens. The test's positive and negative predictive values were also high (95 and 86%, respectively). In conclusion, a sensitive and specific immunoassay was developed for the detection of HpCA in human serum. This test can be applied for noninvasive laboratory and field diagnoses of H. pylori infection.     

Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis

Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence of Cryptosporidium infection in the population.     

Incorporation of Different Bridge Length Linkers in Enzyme and Its Use in the Preparation of Enzyme Conjugates for Immunoassay

Abstract

An enzyme horseradish peroxidase (HRP), as a starting material, has been used to introduce different bridge length linkers, and its use in the preparation of enzyme conjugates for immunoassay is described. HRP was conjugated to adipic acid dihydrazide (ADH), gamma amino butyric acid (GABA), followed by ADH and 6‐amino caproic acid (6ACA) followed by ADH. The different bridge length linkers‐incorporated enzyme was coupled to a carboxylic derivative of cortisol. Four enzyme conjugates with different bridge length were prepared, such as cortisol‐21‐hemisuccinate–HRP (cortisol‐21‐HS–HRP), cortisol‐21‐HS–ADH–HRP, cortisol‐21‐HS–ADH–GABA–HRP, and cortisol‐21‐HS–ADH–6ACA–HRP. The influence of linker on sensitivity and specificity of the cortisol assay was studied. The study revealed that incorporation of a linker between hapten and enzyme increases the sensitivity and specificity of the assay.     

Utility of Galactomannan Enzyme Immunoassay and (1,3) β-d-Glucan in Diagnosis of Invasive Fungal Infections: Low Sensitivity for Aspergillus fumigatus Infection in Hematologic Malignancy Patients

Previous studies have reported that galactomannan (GM) enzyme immunoassay and 1,3 beta-glucan (BG) assay may be useful diagnostic tools, but their sensitivities are variable. We compared the performances of both tests. Between October 2002 and May 2005, 82 patients were prospectively monitored for 12 weeks. A total of 414 samples were tested by GM assay and 409 samples were tested by BG assay for the following four groups of patients: those with invasive aspergillosis (IA), those with other mold infections (Fusarium, scedosporium, zygomycosis, etc.), those with candidemia, and control patients. Blood samples were obtained twice on week 1 and once every other week for a total of 12 weeks. Patients in the invasive fungal infection groups had comparable risk factors. The sensitivity of the GM test was significantly higher for patients with IA due to non-fumigatus Aspergillus species than for patients with IA due to Aspergillus fumigatus (49% versus 13%; P < 0.0001) or with other mold infections (49% versus 6%; P < 0.0001). However, the sensitivity range (47% to 64%) and specificity (88%) of the BG assay were comparable among all patients tested, regardless of the infecting pathogen. The performance of GM-based diagnosis appears to be better for detecting non-fumigatus Aspergillus species. The diagnostic marker BG was shown to have a higher sensitivity than that of GM in detecting IA and other mold infections in hematologic malignancy patients.     

Use of Protein a Hearing Staphylococcus Aureus in Enzyme Immunoassay (EIA)

Bovine serum albumin labeled with alkaline phosphatase and antibody have been employed as a model to determine if the use of Protein A bearing Staphylococcus aureus Cowan I strain (SACl) bacteria could be extended to enzyme immunoassay (EIA). SACl do not activate “per se” the enzyme substrate and bind aspecifically minimum amount of enzyme labeled antigen. Experimental conditions are described for the use of SACI both in macro and micro EIA assay which allows the processing of numerous samples with minimum hand ling. The sensitivity of the EIA is comparable with radioassay (EIA 2ng-RIA 4ng) which uses SACI in place of second antibody. The inhibition test can be performed in 4 hours time. These results suggest that the stability of SACl when combined with that of enzyme labeled antigens can widen the use of EIA, both for investigative and clinical studies.     

Use of Protein a Hearing Staphylococcus Aureus in Enzyme Immunoassay (EIA)

Bovine serum albumin labeled with alkaline phosphatase and antibody have been employed as a model to determine if the use of Protein A bearing Staphylococcus aureus Cowan I strain (SACl) bacteria could be extended to enzyme immunoassay (EIA). SACl do not activate “per se” the enzyme substrate and bind aspecifically minimum amount of enzyme labeled antigen. Experimental conditions are described for the use of SACI both in macro and micro EIA assay which allows the processing of numerous samples with minimum hand ling. The sensitivity of the EIA is comparable with radioassay (EIA 2ng-RIA 4ng) which uses SACI in place of second antibody. The inhibition test can be performed in 4 hours time. These results suggest that the stability of SACl when combined with that of enzyme labeled antigens can widen the use of EIA, both for investigative and clinical studies.     

Cross-Reactivity of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Cryptococcus Species in the Commercial Platelia Aspergillus Enzyme Immunoassay

Cross-reactivity in the Platelia Aspergillus enzyme immunoassay was evaluated using 120 sera from patients with paracoccidioidomycosis, histoplasmosis, and cryptococcosis. At a cutoff value of 0.5, positivity rates were 50%, 67%, and 50%, respectively. The implications for these findings are discussed.     

Cross-reactivity of Fusarium spp. in the Aspergillus Galactomannan enzyme-linked immunosorbent assay

Nine of 11 hematological patients with disseminated/deep-seated Fusarium infection tested at least twice for Aspergillus galactomannan (GM) had repeated positive results in the absence of Aspergillus isolation in culture. The centrifuged supernatants of 12 Fusarium isolates were tested by a GM enzyme-linked immunosorbent assay (EIA). All the isolates produced positive reactions when tested undiluted. These results show cross-reactivity of Fusarium spp. with Aspergillus GM that may constitute a drawback with respect to the specificity of the Platelia EIA.     

Causes of Outbreaks Associated with Drinking Water in the United States from 1971 to 2006

Summary: Since 1971, the CDC, EPA, and Council of State and Territorial Epidemiologists (CSTE) have maintained the collaborative national Waterborne Disease and Outbreak Surveillance System (WBDOSS) to document waterborne disease outbreaks (WBDOs) reported by local, state, and territorial health departments. WBDOs were recently reclassified to better characterize water system deficiencies and risk factors; data were analyzed for trends in outbreak occurrence, etiologies, and deficiencies during 1971 to 2006. A total of 833 WBDOs, 577,991 cases of illness, and 106 deaths were reported during 1971 to 2006. Trends of public health significance include (i) a decrease in the number of reported outbreaks over time and in the annual proportion of outbreaks reported in public water systems, (ii) an increase in the annual proportion of outbreaks reported in individual water systems and in the proportion of outbreaks associated with premise plumbing deficiencies in public water systems, (iii) no change in the annual proportion of outbreaks associated with distribution system deficiencies or the use of untreated and improperly treated groundwater in public water systems, and (iv) the increasing importance of Legionella since its inclusion in WBDOSS in 2001. Data from WBDOSS have helped inform public health and regulatory responses. Additional resources for waterborne disease surveillance and outbreak detection are essential to improve our ability to monitor, detect, and prevent waterborne disease in the United States.     

Analysis of Eight Commercial Enzyme Immunoassay Tests for Detection of Antibodies to Mycoplasma pneumoniae in Human Serum

Mycoplasma pneumoniae is an important etiologic agent of primary atypical pneumonia in children and adults. The diagnosis of M. pneumoniae infection is commonly confirmed through serologic testing. In this study, we used paired sera from 51 patients (all with confirmed M. pneumoniae infection and positive complement fixation [CF] titers) to compare the results of eight enzyme immunoassays (EIAs) available commercially in the United States. We compared two single-use EIAs and six plate-type EIAs. Results from acute-phase sera ranged from only 7 (14%) positive by ImmunoWELL (GenBio) immunoglobulin M (IgM) EIA to 23 (45%) positive by Zeus IgG EIA. When both the acute-phase and convalescent-phase serum samples were examined, positive results ranged from 20 (39%) by the ImmunoWELL (GenBio) IgM assay to 45 (88%) positive by the Remel IgG-IgM EIA. In this study, the single-use EIAs by Remel and Meridian were more reliable than were the plate-type EIAs. Among the plate-type EIAs, the Zeus and DiaSorin assays (which detect antibodies to protein antigens) were more sensitive than the ImmunoWELL assay (which detects antibodies to glycolipid antigens). In general, IgG EIAs on convalescent-phase sera were more concordant with one another than were IgM EIAs with one another. Scatter plot analysis of convalescent-phase sera showed that, as the CF titer dropped, the IgM assays identified fewer positive convalescent-phase sera. In contrast, the IgG assays provided fairly consistent positive results for convalescent-phase sera with CF titers of 64 and above. Results of individual tests and overall limitations of serodiagnostics for M. pneumoniae infections are discussed.     

高灵敏均相酶免疫法测定血清皮质醇方法建立与性能评价

目的 建立高灵敏均相酶免疫法定量检测血清皮质醇.方法 对此方法的线性范围、准确度、精密度、抗干扰能力、临床可报告范围以及参考区间进行性能验证.结果 本研究建立的血清皮质醇均相酶免疫测定方法,线性范围为30.0～1200.0ng/mL,准确度相对偏差B≤10.0％,批内CV≤10.0％,批间差R≤10.0％,当样本中胆红素≤50mg/L、血红蛋白≤1000mg/L、白蛋白≤100g/L、抗坏血酸≤176mg/dL时,对测定结果无影响,临床可报告范围上限6500ng/mL,血清皮质醇参考区间为60～230ng/mL.结论 该均相酶免疫法能够满足临床血清皮质醇检测的需求,可以进一步推广至临床使用.     

Application of a Timing Protocol to the Reduction of Inter-Plate Variability in the Indirect Enzyme Immunoassay for Detection of Anti-Brucella Antibody

A simple timing protocol was developed to monitor chromogen conversion in an enzyme immunoassay, performed in microtiter plates, for the detection of antibody to Brucella abortus in bovine serum. Application of this protocol decreased the inter-plate coefficient of variation from 28.6% to 6.8% when optical density (OD) values, subsequent to the reaction of a standard antibody reagent, were compared to a static development time. Substantial reductions in variation were also observed for low titered seropositive and for seronegative control reagents. The timing protocol was based on the mathematical relationship of the OD value at 4 minutes of development to a predetermined target OD value (1.0) for a standard antibody reagent. Application of this relationship to the calculation of a variable, final development time eliminated the need for extensive data manipulation and assay calibration.