Detection of free-living amoebae in domestic cats with and without naturally-acquired keratitis.

Pathogenic free-living amoebae, most notably Acanthamoeba spp., are important pathogens of the human cornea. The importance of infection with free-living amoebae in cats with keratitis is currently unclear. The aim of this study was to determine the frequency of amoeba detection in corneas of cats with naturally-acquired keratitis and in the ocular surface microflora of cats without ocular disease. Clinical ophthalmic and in vivo corneal confocal microscopic examinations were performed on 60 cats with keratitis. Corneal scrapings were analyzed by amoeba culture; cytological evaluation; and Acanthamoeba, Hartmannella, and Vahlkampfia PCR assays. Following ophthalmic examination, conjunctival specimens collected from 60 cats without clinically apparent ocular disease were analyzed similarly. In one cat with ulcerative keratitis, amoeba cysts and trophozoites were detected by in vivo corneal confocal microscopy; an Acanthamoeba sp. was isolated from corneal specimens and detected by Acanthamoeba PCR assay; and suppurative corneal inflammation was present cytologically. An Acanthamoeba sp. was isolated from conjunctival specimens from one cat without clinically apparent ocular disease, but with suppurative inflammation demonstrated cytologically. Both Acanthamoeba isolates belonged to the T4 genotype. Naegleria-like amoebae were isolated in samples from two cats with keratitis and seven cats without clinical ocular disease, but amoebae were not detected by the other assays in these samples. Amoeba detection by culture was significantly (P = 0.01) associated with cytologically diagnosed corneoconjunctival inflammation. This study identified naturally-acquired Acanthamoeba keratitis in cats. Detection of Naegleria-like amoebae in samples from cats with and without keratitis is of uncertain pathological significance.

Cryptosporidium parvum: determination of ID50 and the dose-response relationship in experimentally challenged dairy calves.

The objectives were to determine the median infective dose (ID50) of Cryptosporidium parvum and to describe the dose-response relationship including associated clinical illness in experimentally challenged dairy calves. Within the first 24h of life, 27 test calves were experimentally challenged with C. parvum oocysts and 3 control calves were sham dosed. Test calves received 1 of 8 possible doses (25, 50, 100, 500, 1 × 10(3), 1 × 10(4), 1 × 10(5), and 1 × 10(6) oocysts). All 27 test calves developed diarrhea. Fecal oocyst shedding occurred in 25 (92.6%) test calves and in 0 control calves. The 2 non-shedding test calves both received 25 oocysts. There was an inverse relationship between dose and time to onset of fecal oocyst shedding (P=0.005). There was no relationship found between dose and duration (P=0.2) or cessation (P=0.3) of fecal oocyst shedding. In addition, there was not a significant relationship between log-dose and the log-peak oocysts (P=0.2) or log-total oocysts (P=0.5) counted/g of feces across the dose groups. There was a positive dose-response relationship between log-dose and diarrhea (P=0.01). However, when controlling for other factors, such as onset and cessation of fecal oocyst shedding, dose was not a significant predictor of diarrhea (P=0.5). Onset and cessation of fecal oocyst shedding were found to be the best predictors of diarrhea (P=0.0006 and P=0.04, respectively). The ID50 for fecal oocyst shedding was 5.8 oocysts, for diarrhea was 9.7 oocysts, and for fecal oocyst shedding with diarrhea was 16.6 oocysts. Given that the ID50 of C. parvum is far less than would be excreted into the environment by a naturally infected calf, prevention and control of cryptosporidiosis is a formidable challenge.

The effects of age on isotypic antibody responses to Aspergillus fumigatus: implications regarding in vitro measurements.

The demonstration of elevated serum immunoglobulin E (IgE), immunoglobulin G (IgG), and immunoglobulin A (IgA) antibodies to Aspergillus fumigatus (Af) is a valuable aid in the serodiagnosis of allergic bronchopulmonary aspergillosis (ABPA). It is important to recognize cases of ABPA, because repeated untreated episodes of pulmonary infiltrates can result in bronchiectasis and even fatal fibrotic lung disease. Serum samples from patients with ABPA have increased isotypic antibodies to Af when compared with patients with asthma and immediate cutaneous reactivity but in whom ABPA has been excluded. There is little information regarding age-related changes of isotypic antibodies to a specific allergen or in comparison of such changes with age-related changes in total class-specific antibody. We determined IgE-Af, IgG-Af, and IgA-Af and total IgE, IgG, and IgA concentrations in serum samples from children with asthma (ages less than 1 through 5 years, 6 through 10 years, and 11 through 15 years) and compared the results with those in children with APBA and adults with asthma. The use of an indirect amplified ELISA with Af adsorbed to microtiter wells demonstrated that serum samples from children with ABPA have elevated IgE-Af, IgG-Af, and IgA-Af concentrations compared with those in serum samples from children with asthma. The assays were sensitive and highly specific in discriminating ABPA from asthma in children. Isotypic antibodies to Af in children do not demonstrate age-related increases as occur for total serum IgE, IgG, and IgA concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluctuations of serum IgA and its subclasses in allergic bronchopulmonary aspergillosis.

Exacerbations of allergic bronchopulmonary aspergillosis (ABPA) are characterized by chest roentgenographic infiltrates, peripheral blood eosinophilia, and elevation of total serum IgE. Also elevated are serum immunoglobulin antibodies directed against Aspergillus fumigatus, IgE-Af, serum IgG-Af, and serum IgA-Af. We measured serum IgA-Af, IgA1-Af, and IgA2-Af by ELISA to determine whether elevations of IgA-Af occurred before or during an exacerbation (like IgG-Af) or after (like IgE-Af). Ten exacerbations of ABPA were studied in seven patients with an average of 10 serial sera per patient analyzed. We used an indirect amplified ELISA with Af initially sensitizing microtiter wells. A "serologic" rise of immunoglobulin Af was identified when optical densities were twice the baseline sera. Serum IgA-Af was elevated over baseline before (n = 5) and during (n = 1) the time of an exacerbation. Serum IgA1-Af was elevated over baseline before (n = 5) or at the time (n = 5) of an exacerbation in all 10 cases. Serum IgA2-Af was elevated before (n = 2) and during (n = 5) exacerbations. Analogous to total serum IgE and IgG-Af, these experiments demonstrate substantial elevations of serum IgA-Af, IgA2-Af, and particularly, IgA1-Af before or during exacerbations characterized by roentgenographic infiltrates. The data are consistent with a contributory role of IgA-Af in the pathogenesis of ABPA.

Lack of cross-reactivity between IgE to salmon and protamine sulfate.

Immediate type-generalized reactions to protamine sulfate are uncommon but may be fatal. The mechanisms of severe or fatal reactions are unknown in most cases. One theory is that contaminating fish (salmon) proteins present in protamine solutions induce anaphylaxis in salmon-sensitive subjects. A second hypothesis is that protamine interacts with anti-salmon IgE to cause anaphylaxis. We assessed these hypotheses by establishing an indirect amplified enzyme-linked immunosorbent assay (ELISA) for IgE to salmon. Sera obtained from two subjects anaphylactically sensitive to salmon demonstrated high binding to salmon that was not inhibited by preincubation of sera with 500 or 1000 micrograms of protamine or Aspergillus fumigatus. Serum from a patient who experienced anaphylactic shock from protamine was indistinguishable from control sera in the ELISA for IgE to salmon. Anti-protamine IgE could not be demonstrated in separate experiments. The assays prove that 1) serum IgE to salmon is not inhibited by protamine and 2) serum from a patient experiencing a severe reaction to protamine did not contain IgE to salmon or protamine. The experiments do not support the notion that there is cross-reactivity between IgE to salmon and protamine sulfate in the cases evaluated.

Prospective immunologic and clinical study of a population exposed to hexamethylene diisocyanate.

We have prospectively evaluated 150 workers exposed to hexamethylene diisocyanate (HDI) and its trimer (THDI) during an 18-month period. The evaluation consisted of periodic serum antibody studies and a questionnaire that was designed to attempt to identify symptoms compatible with work-related syndromes of allergic rhinitis, allergic conjunctivitis, hypersensitivity pneumonitis, asthma, or irritant reactions. The study population was divided into seven groups on the basis of job classification. The groups differed in exposure levels but were similar in terms of age, sex, smoking history, and duration of work with isocyanates. IgE and IgG against HDI and THDI conjugated to human serum albumin (HSA) (HDI-HSA and THDI-HSA) were determined by ELISA. There were no instances of immunologically induced disease among the 21% of workers in this sample with antibody; however, there is insufficient evidence at this time to make judgments about the relationship between antibody and clinical disease. The antibody was generally low-level IgG that may be a sensitive indicator to detect exposure to certain reactive chemicals. The level of antibody was not different among job classes or between smokers and nonsmokers. Moreover, there was no correlation between antibody level and exposure duration in these workers whose exposure levels are all well below National Institute for Occupational Safety and Health recommendations. Further evaluation will extend these observations.

Analysis of bronchoalveolar lavage in allergic bronchopulmonary aspergillosis: divergent responses of antigen-specific antibodies and total IgE.

Bronchoalveolar lavage (BAL) was performed in eight patients with allergic bronchopulmonary aspergillosis (ABPA) at a time when chest roentgeongraphy did not reveal an infiltrate, and respiratory status was stable. BAL was tolerated well by all patients with only one patient experiencing mild wheezing. BAL fluid recovery averaged 40%, and total cells/lavage were 22.3 x 10(6) (range 3.5 to 49.5 x 10(6)). Cell viability, as determined by trypan blue exclusion, averaged 48% (range 34% to 60%). Mean values for cellular elements were macrophages, 62%; epithelial cells, 12%; lymphocytes, 16%; neutrophils (PMN), 4%; and eosinophils, 6%. Isotypic antibodies to Aspergillus fumigatus (Af) in BAL and serum were detected by an amplified indirect ELISA. Antibodies to Af in BAL expressed as optical density/albumin (milligrams per milliliter) were compared to BAL from six nonatopic patients. IgE-Af and IgA-Af in BAL were elevated in patients with ABPA compared with six nonatopic patients. The ratios of Ig-Af in BAL to peripheral blood in patients with ABPA were 48 (range 18 to 75) for IgE-Af, 96 (range 37 to 159) for IgA-Af, and 0.94 (range 0.24 to 1.40) for IgG-Af, suggesting local production of IgE-Af and IgA-Af in the bronchoalveolar compartment. Total serum IgE correlated directly with IgE-Af in BAL (rs = 0.67; p less than 0.02). However, the ratio of total BAL IgE/albumin divided by total serum IgE/albumin was 0.93 +/- 0.94, suggesting that the bronchoalveolar compartment is not the source of the significant elevations in total serum IgE in ABPA.

Immune responses to Aspergillus in cystic fibrosis.

Aspergillus fumigatus (Af) is well recognized in its ability to colonize the respiratory tract in cystic fibrosis (CF). Furthermore, a number of the immune responses of the patient with CF to this organism have been characterized, and the immune inflammatory response to Af may result in allergic bronchopulmonary aspergillosis (ABPA). This study evaluated a series of immunologic parameters in 75 patients with CF in order to characterize more fully the spectrum of immune responses of those patients to Af and to clarify the relationship of those responses to the clinical features of ABPA. The patients could be classified into four groups, depending on the clinical and immunologic findings. Eight (10.7%) of the 75 patients had clinical and laboratory evidence of ABPA, including immediate cutaneous reactivity to Af, eosinophilia, elevated total serum IgE, elevated serum IgE-Af or IgG-Af, and precipitating antibody to Af. Ten (13.3%) patients had these features, except that the total serum IgE level was within the normal range. Forty (53.5%) of the patients had no significant criteria for ABPA but had varying immunologic responses to Af, such as immediate cutaneous reactivity to Af in 25 patients and elevated serum IgE-Af and/or IgG-Af in 19 patients. Seventeen (22.7%) patients had no evidence of an immunologic response, as determined by skin testing and serologic assays. The study demonstrated that the response of patients with CF to Af ranges from clinically apparent ABPA to a possible variant of ABPA, to a nondiagnostic group of features consistent with sensitization to Af or to no characteristic immune response.

Allergic bronchopulmonary candidiasis: case report and suggested diagnostic criteria.

A patient with an illness consistent with allergic bronchopulmonary candidiasis is described. The patient had asthma, atelectatic pulmonary infiltrates on three occasions, immediate cutaneous reactivity as low as 10(-7) (wt/vol) to Candida albicans extract, and precipitating antibody to this organism. C. albicans was the only organism cultured from two bronchial lavage specimens. Total serum IgE was elevated to 5745 ng/ml and decreased rapidly with corticosteroid therapy. Serologic studies were not consistent with allergic bronchopulmonary aspergillosis. Serum IgE to C. albicans, measured by ELISA after adsorption of IgG from the serum samples by incubation with staphylococcal protein A, was found to be 575% to 650% above control values. The serum IgE antibody activity against Candida decreased with clinical improvement after corticosteroid therapy.

Prolonged evaluation of patients with corticosteroid-dependent asthma stage of allergic bronchopulmonary aspergillosis.

Eight cases with stage IV allergic bronchopulmonary aspergillosis (ABPA) (corticosteroid-dependent asthma stage) were observed for a total of 82 patient years with individual patients observed for 7 to 19 years (mean 10.2) years. One case is the first case of ABPA diagnosed in the United States in 1967. A second case has been observed through four stages of ABPA. None of these eight cases has demonstrated pulmonary deterioration by clinical, chest roentgenogram, or pulmonary function analysis. After diagnosis, the maintenance dose of prednisone in seven of eight cases was a low to moderate dose alternate-day prednisone. These results suggest that continuous observation and management of episodes of pulmonary consolidation or asthma exacerbations may prevent the progression of ABPA to stage V (fibrotic end stage). The total IgE may remain elevated in these patients, and therapy should not attempt to reduce total serum IgE to normal levels. After prolonged therapy with prednisone for asthma and control of ABPA, the IgE and IgG antibody indices against Aspergillus fumigatus may remain elevated or may be below the levels that are of diagnostic value.

Stage V (fibrotic) allergic bronchopulmonary aspergillosis. A review of 17 cases followed from diagnosis.

A review of the records of 17 patients with stage V (fibrotic stage) allergic bronchopulmonary aspergillosis observed since initial diagnosis (mean observation period, 4.9 years) demonstrated that, of the 11 surviving patients, four have very severe respiratory impairment. The other seven patients have mild or moderate functional impairment, but most of these have not shown clinical deterioration during the observation period. The occurrence of new roentgenographic infiltrates after the time of diagnosis was observed in only one patient in this series. Serum IgE and IgG levels against Aspergillus fumigatus, when compared with those of a control pool of serum samples from asthmatic patients with immediate cutaneous reactivity to Aspergillus, were the most useful immunologic studies diagnostically. Lung biopsy specimens obtained in five patients were of relatively little diagnostic value. All patients have required long-term prednisone therapy for control of asthma. Those patients whose forced expiratory volume in 1 s (FEV1) remained less than or equal to 0.8 L after initial corticosteroid treatment demonstrated a poor prognosis. When only moderate lung damage has occurred at the time of diagnosis, a stable subsequent course may be expected even in patients with stage V disease.

Serum IgA antibodies to Aspergillus fumigatus in various stages of allergic bronchopulmonary aspergillosis.

With double antibody ELISA, serum IgA antibodies against Aspergillus fumigatus (Af) were measured from patients in stages I through V allergic bronchopulmonary aspergillosis (ABPA). All sera from patients with ABPA demonstrated considerably greater values for IgA-Af than sera from nonatopic subjects. When this sera was compared with sera from patients with asthma and immediate cutaneous reactivity to Aspergillus, the present studies documented markedly elevated serum IgA-Af in the acute, exacerbation, and fibrotic stages of ABPA. Some patients in remission or corticosteroid-dependent asthma stages also demonstrated increased values. Because the major stimulus to antibody production occurs in the lung in response to presence of Af hyphae, polymeric IgA antibodies could potentially contribute to immunologic lung damage in ABPA. Finally, large dose alternate day or daily prednisone that was administered to patients in the fibrotic stage of ABPA did not prevent the marked production of isotypic antibodies to Af.

Allergic bronchopulmonary aspergillosis. Natural history and classification of early disease by serologic and roentgenographic studies.

Eighty-four patients with allergic bronchopulmonary aspergillosis (ABPA) were evaluated for a total of 294 patient-years with a mean observation period of 3.7 years and classified by the stage of ABPA. The largest percentage of patients were in the stage IV (corticosteroid-dependent asthma stage) group. The next largest percentage were in the stage V (fibrotic, end-stage lung disease) group. Of the latter 24 patients, eight had died. In addition, we describe 13 patients with all serologic characteristics of ABPA but without central bronchiectasis. We propose that these patients have seropositive ABPA and represent the earliest cases of it that can be diagnosed in contrast with ABPA with central bronchiectasis in which lung damage is already present.