Allergic Bronchopulmonary Mycosis due to fungi other than Aspergillus.

Summary: Allergic bronchopulmonary mycosis (ABPM) is a clinical syndrome associated with immune sensitivity to various fungi that colonize the airways. Early diagnosis and treatment with systemic corticosteroids is the key in preventing the progression of the disease to irreversible lung fibrosis. Although Aspergillus has progressively gained recognition as a causative agent in past few decades, other fungi, that have been reported to cause ABPM, are not yet widely evaluated. We studied hundred and two patients with asthma for occurrence of ABPM. Patients were tested for cutaneous hypersensitivity and serum precipitin to 12 common fungal antigens. The positive cases were further evaluated for ABPM using standard criteria. Out of 102 asthma patients screened, 18 patients had either skin prick test (SPT) and/or serum precipitin positive. While 14 patients were SPT positive for one or more fungal antigen, two patients were serum precipitin positive for one or more fungi. Two patients had both serum precipitin positive as well as SPT positive. Six (5.8%) patients were diagnosed as ABPM as they fulfilled the criteria. Three of these were because of Aspergillus sp. Two were because of fungi other than Aspergillus namely Schizophyllum and Curvularia. One patient had ABPM because of both Aspergillus and Curvularia. In our study absolute eosinophil count (AEC), total IgE, serum precipitin and SPT had sensitivity of 100%, 100% 50% and 83.3% respectively for diagnosing ABPM. The specificity of these tests was 44.79%, 64.58% 98.96% and 88.54% respectively. Specfic IgE was positive in 50% of patients with either serum precipitin or SPT positivity. SPT or serum precipitin followed by specific IgE had sensitivity of 100% and specificity of 96.88% for diagnosing ABPM. SPT alone followed by Specific IgE had a sensitivity of 83.33% and specificity of 96.88% for diagnosing ABPM. We found that fungi other than Aspergillus such as schizophyllum, and curvularia, can be implicated in ABPM. Multiple fungal agents may be responsible for ABPM in an individual. There is a subset of patients of BA who have fungal sensitization but do not fulfil the criteria for ABPM. SPT was the single most sensitive and specific test, AEC >350 and total IgE more than 417IU were most sensitive tests and SPT followed by specific IgE was most effective strategy for diagnosing ABPM.

Diagnostic implications of positive avian serology in suspected hypersensitivity pneumonitis.

BACKGROUND: The diagnostic evaluation of patients with interstitial lung disease (ILD) often involves serologic assessment for identifiable causes such as hypersensitivity pneumonitis (HP). While not on its own defining of HP, precipitin serologies are often obtained to support clinical suspicion if other findings are inconclusive. We studied the clinical relevance of positive avian serology in patients undergoing ILD evaluation. MATERIAL AND METHODS: We identified individuals with positive avian serology (>53.3 mg/L) and undifferentiated ILD seen at our institution over a three-year period. Clinical, laboratory, pathologic, and radiologic findings were evaluated for consensus HP diagnosis by two expert pulmonologists, blinded to presenting serology levels. RESULTS: Ninety-one ILD subjects with positive avian serology were identified; mean age was 62.7 ± 15.3 years with a slight male predominance (56%). Forty-nine (54%) received a consensus HP diagnosis. Those with HP had higher mean avian serology titer (95.0 ± 38.7 mg/L vs. 68.3 ± 16.7, (P < 0.0001). Never-smokers also had higher titers compared to prior or active smokers (P = 0.0008). Positive avian protein exposure (P < 0.0001, OR 21.3 (6.4-87)), DLCO% (P = 0.04, unit OR 0.96 (0.92-0.99)), and increasing serology titer (P < 0.015, unit OR 1.03 [1.01-1.06]) were independent predictors of HP diagnosis. CONCLUSION: Among patients with positive avian serology, those with higher titers were more likely to have HP diagnosis. Nonsmokers also manifested higher titers compared to those with smoking history. These results may guide the usage and interpretation of avian serology screening in the initial assessment of suspected HP.

Comparative Study of the Accuracy of Different Techniques for the Laboratory Diagnosis of Schistosomiasis Mansoni in Areas of Low Endemicity in Barra Mansa City, Rio de Janeiro State, Brazil.

Schistosomiasis constitutes a major public health problem, with an estimated 200 million people infected worldwide. Many areas of Brazil show low endemicity of schistosomiasis, and the current standard parasitological techniques are not sufficiently sensitive to detect the low-level helminth infections common in areas of low endemicity (ALEs). This study compared the Kato-Katz (KK); Hoffman, Pons, and Janer (HH); enzyme-linked immunosorbent assay- (ELISA-) IgG and ELISA-IgM; indirect immunofluorescence technique (IFT-IgM); and qPCR techniques for schistosomiasis detection in serum and fecal samples, using the circumoval precipitin test (COPT) as reference. An epidemiological survey was conducted in a randomized sample of residents from five neighborhoods of Barra Mansa, RJ, with 610 fecal and 612 serum samples. ELISA-IgM (21.4%) showed the highest positivity and HH and KK techniques were the least sensitive (0.8%). All techniques except qPCR-serum showed high accuracy (82-95.5%), differed significantly from COPT in positivity (P < 0.05), and showed poor agreement with COPT. Medium agreement was seen with ELISA-IgG (Kappa = 0.377) and IFA (Kappa = 0.347). Parasitological techniques showed much lower positivity rates than those by other techniques. We suggest the possibility of using a combination of laboratory tools for the diagnosis of schistosomiasis in ALEs.

Comparative study of the accuracy of different technique for the laboratory diagnosis of schistosomiasis mansoni in areas of low endemicity in Barra Mansa City, Rio de Janeiro State, Brazil

Schistosomiasis constitutes a major public health problem, with an estimated 200 million people infected worldwide. Many areas of Brazil show low endemicity of schistosomiasis, and the current standard parasitological techniques are not sufficiently sensitive to detect the low-level helminth infections common in areas of low endemicity (ALEs). This study compared the Kato-Katz (KK); Hoffman, Pons, and Janer (HH); enzyme-linked immunosorbent assay- (ELISA-) IgG and ELISA-IgM; indirect immunofluorescence technique (IFT-IgM); and qPCR techniques for schistosomiasis detection in serum and fecal samples, using the circumoval precipitin test (COPT) as reference. An epidemiological survey was conducted in a randomized sample of residents from five neighborhoods of Barra Mansa, RJ, with 610 fecal and 612 serum samples. ELISA-IgM (21.4%) showed the highest positivity and HH and KK techniques were the least sensitive (0.8%). All techniques except qPCR-serum showed high accuracy (82­95.5%), differed significantly from COPT in positivity , and showed poor agreement with COPT. Medium agreement was seen with ELISA-IgG (Kappa = 0.377) and IFA (Kappa = 0.347). Parasitological techniques showed much lower positivity rates than those by other techniques. We suggest the possibility of using a combination of laboratory tools for the diagnosis of schistosomiasis in ALEs.

Detection of Schistosoma mansoni antibodies in a low-endemicity area using indirect immunofluorescence and circumoval precipitin test.

Parasitological diagnostic methods for schistosomiasis lack sensitivity, especially in regions of low endemicity. The objective of this study was to determine the prevalence of Schistosoma mansoni infections by antibody detection using the indirect immunofluorescence assay (IFA-IgM) and circumoval precipitin test (COPT). Serum samples of 572 individuals were randomly selected. The IFA-IgM and COPT were used to detect anti-S. mansoni antibodies. Of the patients studied, 15.9% (N = 91) were IFA-IgM positive and 5.1% (N = 29) had COPT reactions (P < 0.001 by McNemar's test). Immunodiagnostic techniques showed higher infection prevalence than had been previously estimated. This study suggests that combined use of these diagnostic tools could be useful for the diagnosis of schistosomiasis in epidemiological studies in areas of low endemicity.

Evaluation of 'cattle' and 'Indian Bison' type antigens of Mycobacterium avium subspecies paratuberculosis for diagnosis of bovine Johne's disease using 'indigenous ELISA' and AGPT.

Two antigens ('cattle' type and 'Indian Bison' type) of Mycobacterium avium subspecies paratuberculosis were evaluated for diagnosis of Johne's disease (JD) in a gaushala (cattle herd). Of the 160 cows of Sahiwal and Hariana breeds screened, 81 (50.6%) tested positive in ELISA and 66 (41.8%) in AGPT test. Using the two antigens, 33.5% tested positive in both the tests while 41.1% tested negative. Exclusively, only 8.2% tested positive in ELISA while 17.1% tested positive in AGPT. Two antigens together detected 58.9% prevalence of MAP in the gaushala. Individually, indigenous ELISA using antigen from native source of MAP proved superior to AGPT in the diagnosis of JD in cows.

Performance of two Aspergillus IgG EIA assays compared with the precipitin test in chronic and allergic aspergillosis.

Detection of Aspergillus IgG antibodies is important in the diagnosis of chronic pulmonary aspergillosis and allergic bronchopulmonary aspergillosis. Immunoprecipitation techniques to detect these antibodies appear to lack sensitivity and accurate quantitation compared with enzyme immunoassays (EIA). This study assessed the performance of two commercial EIAs compared with counterimmunoelectrophoresis (CIE). This was a prospective cohort study of 175 adult patients with chronic or allergic pulmonary aspergillosis. Aspergillus IgG antibodies were detected using CIE, Phadia ImmunoCap Aspergillus IgG and Bio-Rad Platelia Aspergillus IgG. Inter-assay reproducibility was determined for each method and 25 patients had two serum samples analysed within a 6-month interval. When compared with CIE, both ImmunoCap and Platelia Aspergillus IgG had good sensitivity (97 and 93%, respectively) for detection of Aspergillus IgG antibodies. The level of agreement between the two EIAs for positive results was good, but the concentration of antibodies was not correlated between the tests or with CIE titre. ImmunoCap IgG inter-assay coefficient of variation was 5%, whereas Platelia IgG was 33%. Median ImmunoCap IgG values for CPA and allergic aspergillosis were 95 and 32 mg/L, respectively, whereas Platelia IgG values were >80 and 6 AU/mL. The direction of CIE titre change over 6 months was mirrored by ImmunoCap IgG levels in 92% of patients, and by Platelia IgG in 72% of patients. Both ImmunoCap and Platelia Aspergillus IgG EIAs are sensitive measures of Aspergillus IgG antibodies compared with CIE. However, ImmunoCap appears to have better reproducibility and may be more suitable for monitoring patient disease.

[Aspergillus serology, from yesterday to today for tomorrow]. / Sérologie aspergillaire, d'hier à aujourd'hui pour demain.

Anti-Aspergillus antibody detection has been performed for over 50 years for the diagnosis of different chronic Aspergillus infections, starting with aspergilloma and later with chronic necrotizing pulmonary aspergillosis. It also enters into definition criteria for allergic broncho-pulmonary aspergillosis and contributes to the initial diagnosis of the aspergillosis, to the follow-up under treatment or to the detection of exacerbations. For the acute invasive aspergillosis, antibody detection has low interest compared to galactomannan antigen detection. Serology results have to be interpreted together with other clinical, radiological and biological, mycological criteria. This review describes the origins, the technical evolutions and the current place of Aspergillus serology in France. Finally, future improvements are discussed.

Detection of antisynthetase syndrome in patients with idiopathic interstitial pneumonias.

OBJECTIVES: Antisynthetase syndrome (ASS) is characterized by autoantibodies to aminoacyl-tRNA synthetases (anti-synthetase) and it is frequently associated with interstitial lung disease. The purpose of this study was to elucidate the prevalence and characteristics of the anti-synthetase positive subpopulation among idiopathic interstitial pneumonias (IIPs) and to clarify the importance of screening for these antibodies. METHODS: A retrospective study was performed in 198 consecutive cases with IIPs. Screening for six anti-synthetase antibodies was performed in all cases. Clinical profiles of all cases were compared with reference to the presence of anti-synthetase. High-resolution computed tomography (HRCT) findings of anti-synthetase positive cases were also analyzed. RESULTS: 13 cases (6.6%) were positive for anti-synthetase. Anti-EJ was most prevalent, followed by anti-PL-12. Onset ages of anti-synthetase positive cases were younger than those of anti-synthetase negative cases. Extrapulmonary features of ASS were absent in 6 anti-synthetase positive cases (46.2%). Histologically, among 5 UIP with lymphoid follicles and 11 NSIP cases, the prevalence of anti-synthetase positive cases was 8/16 (50%). On HRCT, ground glass opacity and traction bronchiectasis were the major findings in anti-synthetase positive cases, while honeycombing was absent. CONCLUSIONS: Anti-synthetase positive cases were not rare among IIPs. Anti-synthetase should be screened for in IIPs, especially in pathological NSIP or UIP with lymphoid follicles. These patients should be screened for anti-synthetase even if no suggestive extrapulmonary manifestation exists.

The utility of polymerase chain reaction for diagnosis of lumpy skin disease in cattle and water buffaloes in Egypt.

An outbreak of lumpy skin disease (LSD) occurred among cattle and water buffaloes in Egypt in 2006. Polymerase chain reaction (PCR) and the agar gel precipitation test (AGPT) were compared. Eight of ten (80%) tissue specimens from diseased cattle were positive with AGPT while 100% were positive with PCR. Of ten tissue specimens from diseased water buffaloes, 70% were positive with AGPT while 100% were positive with PCR. Ten milk samples were obtained from diseased water buffaloes; PCR detected nucleic acid of LSD virus (LSDV) in 50% while AGPT failed to detect LSDV antigen. Water buffaloes are susceptible to LSDV infection. The clinical signs of LSD were less severe in water buffaloes, but the virus was excreted in their milk. Diagnosis of LSD outbreaks by PCR will facilitate rapid application of control measures. Mass vaccination should be applied in both cattle and water buffaloes in Egypt using an effective specific vaccine against LSD, such as the attenuated Neethling strain vaccine or a recombinant vaccine.

Comparison of Aspergillus galactomannan antigen testing with a new cut-off index and Aspergillus precipitating antibody testing for the diagnosis of chronic pulmonary aspergillosis.

BACKGROUND AND OBJECTIVE: The usefulness of two tests in the serodiagnosis of chronic pulmonary aspergillosis (CPA) was compared. The tests were the serum Aspergillus galactomannan antigen test (Platelia (R) Aspergillus) by enzyme-linked immunoassay (EIA) using old and new cut-off indexes, and the Aspergillus precipitating antibody test. METHODS: Both Aspergillus-precipitating antibody and Platelia Aspergillus EIA positivity were measured in the sera of 28 patients at the time of diagnosis of CPA. RESULTS: Serum Aspergillus precipitating antibody positivity was 89.3% (25/28) in CPA patients. Serum Platelia Aspergillus EIA positivity was 21.4% (6/28) using the old cut-off index (> or =1.5) and 50% (14/28) using the new cut-off index (> or =0.5)-still less than that for Aspergillus precipitating antibody. Three of the 28 CPA patients had positive reactions in the Platelia Aspergillus EIA using the old cut-off index but not in the Aspergillus precipitating antibody test. Positivity for (1,3) beta-d glucan was 15.4%, and that for culture on CHROMagar Candida was 17.9%. One patient with pulmonary actinomycosis had a false-positive reaction in the Platelia Aspergillus test with the new cut-off index. CONCLUSIONS: For the diagnosis of CPA, Aspergillus precipitating antibody testing is more sensitive than the Platelia Aspergillus EIA, even with the new cut-off index. False-positive reactions are observed with the Platelia Aspergillus EIA in patients with conditions such as pulmonary actinomycosis. Results should be interpreted with care when patients are positive for the Platelia Aspergillus EIA but negative for Aspergillus precipitating antibody.

Comparative efficacy of standard AGID and precipitinogen inhibition test with monoclonal antibodies based competitive ELISA for the serology of Peste des Petits Ruminants in sheep and goats.

This project was conducted to investigate the comparative efficiency of competitive ELISA (cELISA), standard Agar Gel Immunodiffusion Test (AGID) and Precipitinogen Inhibition Test (PIT) for the diagnosis of Peste des Petits Ruminants (PPR) in Pakistan. To deal with this, serum samples from 198 sheep and 82 goats were collected from three different government livestock farms and all the samples were run simultaneously with the three serological tests. The samples found positive for PPR antibodies through cELISA, AGID and PIT were 96 (34.2%), 60 (21.4%) and 72 (25.7%), respectively. Kappa statistics were applied to evaluate the concordance between the laboratory-based test (cELISA) and field-based tests (AGID and PIT). Kappa statistics scores for cELISA versus AGID and PIT were 0.6343 (95% Confidence Interval CI 0.5231-0.7456) and 0.7134 (95% Confidence Interval CI 0.5987-0.8281), respectively, which indicate a "substantial" agreement between cELISA and AGID and "significant" agreement between cELISA and PIT. AGID and PIT revealed relative diagnostic sensitivities with cELISA of 59.3% and 69.7% and relative diagnostic specificities of 98.3% and 97.2%, respectively. The data suggested that for mass screening and control of PPR, these serological tests proved practical in the absence of cELISA since they have high relative diagnostic specificities and a satisfactory relative diagnostic sensitivities.

Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ: effects of GSK3beta activation and caspase 3 cleavage.

To quantitatively measure tau aggregation in situ, we established a cell model system using a split green fluorescence protein (GFP) complementation assay. In this assay the more aggregated the protein of interest the lower the GFP fluorescence. Tau microtubule-binding domain constructs, whose aggregation characteristics have been described previously (Khlistunova et al. 2006), were used to validate the assay. The aggregation-prone construct exhibited the lowest GFP intensity whereas the aggregation-resistant construct showed the highest GFP intensity. To examine the role of glycogen synthase kinase 3beta (GSK3beta) activity and caspase 3 cleavage on tau aggregation, GFP complementation of full length (T4), caspase-cleaved (T4C3), and pseudophosphorylated at S396/S404 (T4-2EC) tau was examined in the presence of an active or a kinase-dead GSK3beta. Extensive phosphorylation of T4 by GSK3beta resulted in increased GFP intensity. T4C3 showed neither efficient phosphorylation nor a significant GFP intensity change by GSK3beta. The GFP intensity of T4-2EC was significantly reduced by GSK3beta accompanying its presence in the sarkosyl-insoluble fraction, thus demonstrating that T4-2EC was partitioning into aggregates. This indicates that if the majority of tau is phosphorylated at S396/S404, in combination with increased GSK3beta activity, tau aggregation is favored. These data demonstrate that split GFP complementation may be a valuable approach to determine the aggregation process in living cells.

Feeding patterns of Haemagogus janthinomys (Diptera: Culicidae) in different regions of Brazil.

New data on the feeding patterns of Haemagogus (Haemagogus) janthinomys Dyar from different geographical regions of Brazil, by using the precipitin test as the bloodmeal-identifying tool, are presented. The following antisera were used: bird, dog, human, rodent, cattle, horse, and opossum. The origins of 287 bloodmeals were identified, whereas 33 specimens were negative to the antiserums tested. Among the reactive specimens, 174 (60.6%) fed on only one food source, of which 35.1% originated from birds, 19.5% from rodents, 12.6% from humans, 10.3% from cattle, 10.3% from opossums, 7.5% from dogs, and 4.6% from horses. One hundred six (37.0%) mosquitoes fed on two sources, of which the most common combinations were bird + rodent (16.0%), bird + human (10.4%), and horse + human (9.4%). Seven (2.4%) mosquitoes fed on three different hosts. Our results suggest that Hg. janthinomys is more eclectic and opportunist than previously known in relation to its hosts and that such patterns are probably highly adaptive to a temporally and spatially variable environment.

Purification of potato virus X and preparation of its antiserum.

Potato virus X (PVX) isolated from the potato leaf and tuber samples which were collected from various fields in Damavand and Ardabil. The initial isolations of the virus were made from potato by mechanical inoculation on Gomphrena globosa L. and Chenopodium spp. that produce local lesion, and then it causes mosaic on Nicotiana spp. and Datura stramonium L. An isolate of the virus inoculated to Nicotiana glutinosa L. and it was maintained throughout the work. Sap from infected N. glutinosa was ineffective after dilution to 10-6, 10 minutes at 70 degrees and 10 weeks at room temperature. The virus was readily purified from infected leaves and the best protocol was Moreira & Jones 1980 than the other 2 methods of Fribourg 1975 and Shepard & Shalla 1972. Antisera were prepared against native, degraded proteins and micro precipitin test showed that both antisera had a 1/512 titer. Precipitin lines with D - Protein antiserum was better of the native protein antiserum in agar double diffusion test than treated with SDS. The isolate of the virus was not transmitted by none of 2 species of Cuscuta but transmitted from infected leaves to healthy plants with sap inoculation without using Carburandum. This isolate showed positive reaction with gamaglubulin in kate received from CIP centre.

Genomic studies with Escherichia coli MelR protein: applications of chromatin immunoprecipitation and microarrays.

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.

Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells.

OBJECTIVE: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. METHODS: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody. RESULTS: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, alpha2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, alpha7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes. CONCLUSIONS: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.