Development of a PCR-based tool for detecting immunologically relevant Alt a 1 and Alt a 1 homologue coding sequences.

Alt a 1 has been recognized as the most important allergen produced by the Pleosporaceae family and is a risk factor for asthma development and/or exacerbation. The aim of this study was to develop a detection tool that is highly specific for species that produced airborne Alt a 1. Based on the highly conserved internal nucleotide region of the several Alt a 1 sequences that are available in GenBank, a pair of primers (Alta1CF/Alta1CR) was designed. A set of primers used by other authors for the production of recombinant Alt a 1 (A21F/A21R) was also tested. The molecular analyses were based on the polymerase chain reaction (PCR) amplification and sequencing of the cDNA that was obtained from thirteen common fungal species. The PCR system that utilized Alta1CF/Alta1CR was highly specific, sensitive, and was able to detect an amplicon of approximately 180 bp from Alt a 1 and Alt a 1-like encoding genes from A. alternata, A. tenuissima, A. infectoria, U. botrytis, and S. botryosum. In contrast, the A21F/A21R primers were specific for the very close taxonomically related species A. alternata and A. tenuissima. Thus, this rapid, sensitive, and specific detection tool can be used to assess Alt a 1 exposure levels and to inform the implementation of the appropriate public health measures.

Diagnostic value of Alt a 1, fungal enolase and manganese-dependent superoxide dismutase in the component-resolved diagnosis of allergy to Pleosporaceae.

BACKGROUND: Over the last decades, genomics and proteomics have contributed to the current knowledge of individualized allergenic components and their potential use in the diagnosis of IgE-mediated allergies. Recent investigations have demonstrated that Alt a 1 should be considered as a relevant allergen of the Pleosporaceae group and that enolase is the main allergen involved in the cross-reactivity to fungi. However, the real utility of these allergens as tools for the diagnosis of allergy to Alternaria is still unclear. OBJECTIVE: To demonstrate the current value of the available fungal allergen panel and the need to build an accurate mould allergen array for the diagnosis of allergy to Pleosporaceae. METHODS: Specific IgEs to individual mould allergens and allergenic mould extracts were evaluated using the ImmunoCAP™ system in 30 patients allergic to Alternaria and in 100 blood donors. Cross-reactivity studies were performed by Fluoro Enzyme ImmunoAssay (FEIA) and FEIA inhibition using individual allergens and allergenic extracts. Two-dimensional electrophoresis associated with a MALDI-TOF analysis was carried out to identify new allergen molecules. RESULTS: All allergic patients had positive specific IgE responses to several moulds from different taxonomical families. Classic and molecular diagnosis demonstrated that 23% of patients had multi-sensitization. The current commercially available fungal allergen array was not sufficient to establish an accurate diagnosis. Unexpected correlations between Alternaria or Alt a 1 and Curvularia or Cladosporium stimulated the investigation of a more accurate allergen panel. A manganese-dependent superoxide dismutase (MnSOD) homologous to Asp f 6 was identified as a new IgE-binding molecule from Alternaria alternata. CONCLUSIONS AND CLINICAL RELEVANCE: Alt a 1 is the marker for allergy to Pleosporaceae, not including Curvularia. MnSOD can explain 6.6% of allergy to Alternaria without Alt a 1 sensitization and should be included together with Alt a 1 and fungal enolase in the molecular array for the diagnosis of allergy to Pleosporaceae.

Alt a 15 is a new cross-reactive minor allergen of Alternaria alternata.

Alternaria alternata is one of the most common saprophytes worldwide that is clinically and epidemiologically associated with severe asthma. Therefore, the identification and characterization of all A. alternata allergens are of major clinical importance. This study describes a new cross-reactive A. alternata allergen that was officially named Alt a 15 by the official Allergen Nomenclature Subcommittee. The complete coding region for Alt a 15 was amplified using 5' and 3' rapid amplification of cDNA ends and PCR. The recombinant protein was produced in Escherichia coli as a 65-kDa fusion protein, and the protein sequence exhibits high homology with several important fungal allergens. Immunoblotting analyses revealed that IgE antibodies from A. alternata-sensitized patients (n=59) bound to rAlt a 15 with a prevalence of 10.2%. All patients who presented sIgE to rAlt a 15 were apparently poly-sensitized to A. alternata and C. lunata. The extensive cross-reactivity between A. alternata and C. lunata serine proteases was confirmed using immunoblotting inhibition assays. Overall, Alt a 15 is an important new cross-reactive allergen of A. alternata that explains some allergies to A. alternata without Alt a 1 sensitization and initial diagnostic errors for allergies to Alternaria. This molecule may improve the accuracy of the diagnosis, the understanding, and the management of IgE-mediated fungal diseases.

Sociodemographic predictors and temporal trends of extrapulmonary tuberculosis as an AIDS-defining disease in Spain.

OBJECTIVE: To identify the factors associated with the presentation of extrapulmonary tuberculosis (EPTB) in AIDS diagnosis and to analyse its temporal trend. DESIGN: Analysis of AIDS surveillance data. METHODS: The study was based on AIDS cases diagnosed in Spain between 1988 and 1993, and reported up to September 1994. The proportion of patients presenting with EPTB at AIDS diagnosis was analysed by sociodemographic characteristics and year of diagnosis. RESULTS: Of the 22,445 AIDS cases diagnosed in Spain from 1988 to 1993, 6526 patients (29.1%; 95% confidence interval, 28.5-29.7) presented with EPTB at diagnosis, making it the most frequent AIDS-defining disease. The highest proportions of EPTB reported at AIDS diagnosis were observed for injecting drug users (IDU; 35.4%) and the heterosexual transmission category (23.5%). The proportion of EPTB was lower in women [relative risk (RR), 0.85; P < 0.001], and higher in 15-29-year-olds (34.2%) and patients with a prison record (44.3 versus 25.4%; RR, 1.75; P < 0.001). AIDS patients resident in provinces with high respiratory TB mortality rates among the general population exhibited a higher proportion of EPTB (RR, 1.45; P < 0.001). The effect of any one variable was maintained by controlling for all others. The proportion of patients with EPTB at AIDS diagnosis revealed a downward trend from 1988 to 1993 (P = 0.007), which was observed in IDU and heterosexuals but not in homosexual/bisexual men (P = 0.421). CONCLUSIONS: Factors such as injecting drug use, prison record or residence in high TB-endemic areas are associated with a greater frequency of EPTB at AIDS diagnosis. These factors must be considered for specific prophylaxis to be efficiently applied.

Changes in AIDS incidence trends in Spain.

OBJECTIVE: To detect changes in the evolution of the AIDS epidemic in Spain. DESIGN: Analysis of time-trends in AIDS incidence. METHODS: AIDS cases reported in Spain up to December 1993 were adjusted for reporting delays. Quarterly time-trends in incidence were analysed for groups divided according to sex, age and transmission category. RESULTS: The adjusted number of cases diagnosed by September 1993 totalled 24,519. Of these, 15,652 were injecting drug users (IDU), 3679 were homosexual/bisexual men and 1753 were cases of heterosexually transmitted infection since 1989. AIDS incidence has exhibited a linear rate of growth. While all groups studied showed an upward trend, variations were found between the different groups. The overall trend resembled the pattern for IDU, the largest but the slope in the growth curve for this category has steepened steadily, and experienced the greatest percentage increases since 1989. AIDS incidence among blood transfusion recipients has risen slowly since 1988, while that for blood factor recipients reached a ceiling in 1989 and has declined progressively thereafter. Within the respective transmission categories, women exhibited trends similar to men. From 1989 onwards, a greater rate of increase was observed among those aged over 30 years than among those under 30. CONCLUSION: The overall trend in AIDS incidence is the sum of the epidemics in the different population groups. While advances in controlling the epidemic have been made in certain groups, the trend as a whole has worsened.

The major allergen of Alternaria alternata (Alt a 1) is expressed in other members of the Pleosporaceae family.

There is general consensus regarding the scarce cross-reactivity existing between Alternaria alternata and other allergenic moulds such as Aspergillus fumigatus, Penicillium notatum or Cladosporium herbarum. However, A. alternata has been shown to have a very significant level of allergenic cross-reactivity with other fungi belonging to the Pleosporaceae family. To date, no biological identity or homologies with other proteins have been described for Alt a 1, and it remains unclear whether the major allergen Alt a 1 contributes to the cross-reactivity shown for these moulds. Specific quantification of Alt a 1 in culture filtrates of Stemphylium botryosum, Ulocladium botrytis, Curvularia lunata, Alternaria tenuissima, C. herbarum, Penicillium chrysogenum and Asp. fumigatus, and immunoblotting using culture filtrate extracts from the above-mentioned moulds and rabbit serum anti-recombinant Alt a 1 have shown significant amounts of Alt a 1 in culture filtrates as well as antigenic components ranging from 14 to 20 kDa that strongly react with the specific serum for all taxonomically related species (Pleosporaceae family). No reactions were revealed in culture filtrates of Cladosporium, Penicillium and Aspergillus. These results restrict the cross-reactivity phenomenon due to Alt a 1 to the scope of the taxonomical term of family.

A case of cardiac hydatidosis.

We present the case of a female patient with a diagnosis of hydatidosis located in the heart. Although echinococcosis is endemic to our country, very few cases of cardiac hydatidosis are normally reported. In our patient, the hydatid cyst was located in the septum and in the right ventricular cavity; it presented other unusual features, such as the fact that it was located exclusively in the heart, that it first manifested as anaphylactic shock of unknown origin and that it required immediate surgical treatment because of severe haemodynamic compromise.

[Estimation of AIDS morbidity and mortality in Spain]. / Estimación de la morbilidad y mortalidad por SIDA en España.

BACKGROUND: To obtain corrected estimates of the cumulative incidence and mortality, as well as prevalence of AIDS in Spain on June 1992. METHODS: The number of cases in the National Register of AIDS by December 1992 was adjusted for reporting delays to estimate the cumulative incidence by June 1992. The prevalence and mortality in this date were obtained using this figure and the probabilities of survival after diagnosis from the AIDS Register of Madrid. This methodology was used for Spain as all, and for each Autonomous Community. RESULTS: The estimated cumulative incidence of AIDS in Spain by June 1992 was 16,486 cases, 13.4% greater than that reported by the same date. The prevalent cases were 6,351 (95% CI, 5,996-6,708) and the remaining 10,135 (61.5%) would have died. This number of deaths is 69.6% greater than the reported figure. There were considerable differences among Autonomous Communities. Some of them exhibited rates more than six times greater than others. CONCLUSIONS: The adjusted estimations provide a view of the actual situation more accurate than the raw figures from the register. These great differences should be taken into account for appropriate allocation of health care resources.