Autoimmunity, phospholipid-reacting antibodies and malaria immunity.

Several questions regarding the production and functioning of autoantibodies (AAb) during malaria infection remain open. Here we provide an overview of studies conducted in our laboratory that shed some light on the questions of whether antiphospholipid antibodies (aPL) and other AAb associated with autoimmune diseases (AID) can recognize Plasmodia antigens and exert anti-parasite activity; and whether anti-parasite phospholipid antibodies, produced in response to malaria, can inhibit phospholipid-induced inflammatory responses and protect against the pathogenesis of severe malaria. Our work showed that sera from patients with AID containing AAb against dsDNA, ssDNA, nuclear antigens (ANA), actin, cardiolipin (aCL) and erythrocyte membrane antigens recognize plasmodial antigens and can, similarly to monoclonal AAb of several specificities including phospholipid, inhibit the growth of P. falciparum in vitro. However, we did not detect a relationship between the presence of anti-glycosylphosphatidylinositol (GPI) antibodies in the serum and asymptomatic malaria infection, although we did register a relationship between these antibodies and parasitemia levels in infected individuals. Taken together, these results indicate that autoimmune responses mediated by AAb of different specificities, including phospholipid, may have anti-plasmodial activity and protect against malaria, although it is not clear whether anti-parasite phospholipid antibodies can mediate the same effect. The potential effect of anti-parasite phospholipid antibodies in malarious patients that are prone to the development of systemic lupus erythematosus or antiphospholipid syndrome, as well as the (possibly protective?) role of the (pathogenic) aPL on the malaria symptomatology and severity in these individuals, remain open questions.

Plasmodium falciparum: limited genetic diversity of MSP-2 in isolates circulating in Brazilian endemic areas.

The genetic polymorphism of the surface merozoite protein 2 (MSP-2) was evaluated in Plasmodium falciparum isolates from individuals with uncomplicated malaria living in a Brazilian endemic area of Peixoto de Azevedo. The frequency of MSP-2 alleles and the survival of genetically different populations clones in 104 isolates were verified by Southern blot and SSCP-PCR. Single and mixed infections were observed in similar frequencies and the rate of detection of FC27 and 3D7 allelic families was equivalent. Eight alleles were identified and among them, the sequence polymorphism was mainly attributed to variations in the repetitive region. Interestingly, in three alleles nucleotide polymorphism was identical to that detected in a previous study, conducted in 1992, in a near Brazilian endemic area. This finding demonstrated the genetic similarity between two isolate groups, besides the certain temporal stability in the allelic patterns. The implications of these data for studies on the genetic diversity are also discussed.

Plasmodium falciparum: a comparative analysis of the genetic diversity in malaria-mesoendemic areas of Brazil and Madagascar.

For a better definition of the polymorphic features of Plasmodium falciparum parasite populations, the polymerase chain reaction (PCR) typing technique was used to investigate the genetic diversity and complexity of parasites harbored by acute P. falciparum carriers from three yet unexplored malaria-mesoendemic areas with different transmission levels: two localities in northwestern Brazil (Ariquemes and Porto Velho) and a village in Madagascar (Ankazobe). A total of 89 DNA samples were analyzed by amplification of polymorphic domains from genes encoding merozoite surface antigens 1 and 2 (MSP-1, MSP-2) and thrombospondin-related anonymous protein (TRAP) and by hybridization with allelic-family-specific probes or random-fragment-length polymorphism (RFLP). In all three localities, extensive polymorphism was observed for each marker, but the MSP-2 central repeat was the most diverse one. Similar levels of genetic diversity, allelic frequency, and infection complexity were observed in the two Brazilian localities, although the isolates had been sampled at 2-year intervals, suggesting the stability of the infecting parasite populations presenting in these regions of the Brazilian Amazon. Unexpectedly, although the entomologic inoculation rate was at least 3 times lower in Ankazobe than in the Brazilian areas. Malagasi samples appeared more complex than the Brazilian ones. The implications of these data with regard to parasite population-dynamics studies are discussed.

Plasmodium berghei: cerebral malaria in CBA mice is not clearly related to plasma TNF levels or intensity of histopathological changes.

Plasmodium berghei ANKA infection in CBA/J mice leads to the development of cerebral malaria (CM) that kills 80-90% of the animals in 6-9 days. This model has been used to study the pathogenesis of CM, which is a major cause of morbidity and mortality in Plasmodium falciparum-infected individuals. The role of cytokines in the induction of CM in the murine model has been well documented, but most studies have been restricted to the peak of neurological manifestations. Here we used a sequential approach to compare mice that developed CM with those that developed no cerebral pathology. Animals were examined for systemic histopathological changes and plasma Tumor Necrosis Factor-alpha (TNF) levels. The objectives were (a) to further determine the importance of factors commonly associated with murine CM-such as elevated levels of TNF and the presence of hemorrhage and vascular plugging-by comparing mice at different stages of infection and/or with different outcomes following infection and (b) to examine the importance of systemic changes-course of parasitemia and histopathological alterations in brain, liver, and lungs-in the development of CM. The data suggest that (a) the clinical manifestation of CM appears to be associated with a wave of merozoite release on days 6-7, (b) murine CM does not present reliable histopathological indicators, (c) there is no topographic association between the occurrence of intravascular plugging and the hemorrhagic foci, (d) monocyte-monocyte and monocyte-endothelial cell adherence were the most expressive histopathological events and were not restricted to brain vessels, (e) blood levels of TNF are not indicative of the local tissue reaction, (f) adhesiveness of monocyte/endothelial cells fluctuate during infection and is dissociated from the lymphocyte homing to the liver, and (g) pulmonary megakaryocytosis (megakaryopoiesis?) is a late event in the lungs.

Cellular and antibody responses to the Plasmodium falciparum heat shock protein Pf72/HSP70 during and after acute malaria in individuals from an endemic area of Brazil.

Proliferative and antibody responses to three synthetic peptides corresponding to Pf72/ HSP70 were followed-up in acute malaria patients from an endemic area of Brazil. In vitro lymphocyte responsiveness to all peptides was relatively low and short-lived and there was a considerable variation in the frequency and magnitude of the individual lymphoproliferative response to the peptides at different periods after the onset of infection. Although 96% of the patients had IgG antibodies to crude Plasmodium falciparum asexual blood stage antigens, specific IgG antibody responses to the peptides varied from 12.5 to 40% according to the tested peptides. No significant difference was observed in the proliferative or antibody responses to the peptides between individuals that remained parasitemic after treatment and those that recovered from malaria infection. The different frequencies of proliferative responses in peripheral blood T cells on different occasions after the onset of their infection show that, in order to be informative, evaluation of the in vitro cellular immune response to peptides requires longitudinal studies in which each individual is tested repeatedly.

A study of antibody and T cell recognition of rhoptry-associated protein-1 (RAP-1) and RAP-2 recombinant proteins and peptides of Plasmodium falciparum in migrants and residents of the state of Rondonia, Brazil.

Humoral and cellular responses were examined among natives and migrants in an area of the Amazon region of Brazil. Rhoptry-associated protein-1 (RAP-1) and RAP-2 expressed in Escherichia coli expression systems, a peptide corresponding to the epitope bound by inhibitory anti-RAP-1 antibodies, and four other RAP-1 and RAP-2 synthetic peptides were used in these studies. Plasma from the native population had greater IgG reactivity to the N-terminal third of RAP-1 than the migrant population; both populations had low levels of IgM to this region of RAP-1. The IgG reactivity to RAP-2 and to the C-terminal third of RAP-1, as well as for all the peptides, including the peptide from the inhibitory domain, were low or absent in both populations. In contrast, there were a high number of subjects with an IgM response to the peptides. Cellular responses were measured by proliferation of peripheral blood mononuclear cells (PBMC) and, in some subjects, by reverse transcription-polymerase chain reaction for interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, and IL-10. Proliferation of PBMC was low when stimulated by recombinant proteins, peptides, or parasite lysate. Both RAP-1 and RAP-2 stimulated cytokine production by donor T cells; IL-2, IL-4, and IFN-gamma RNA transcripts were observed in response to recombinant proteins and parasite lysate, but with no uniform trends. From the observed antibody responses, RAP-1 appears to be more immunogenic than RAP-2.

Seroepidemiology of abdominal angiostrongyliasis: the standardization of an immunoenzymatic assay and prevalence of antibodies in two localities in southern Brazil.

Abdominal angiostrongyliasis is a nematode disease produced by Angiostrongylus costaricensis, a metastrongylid parasite of wild rodents. Accidental human infection occurs through ingestion of food or water contaminated with third-stage larvae present in the mucous secretion of terrestrial molluscs. An ELISA test was standardized for detection of IgG antibodies recognizing a surface antigen prepared from female worms. Competitive absorption of sera with Ascaris suum crude antigen resulted in a test with 86% sensitivity and 83% specificity. The disease is endemic in Southern Brazil and a number of cases are diagnosed every year through anatomo-pathological examination of biopsies or surgical specimens, since no other diagnostic method is available. According to seroepidemiological studies, prevalences in two transmission foci are 29.8 and 66%, attesting to the widespread occurrence of the infection in those endemic areas.

Malaria diagnosis: standardization of a polymerase chain reaction for the detection of Plasmodium falciparum parasites in individuals with low-grade parasitemia.

In Brazil, no study has been done concerning the detection of malaria parasites by polymerase chain reaction (PCR) related to the diagnosis of Plasmodium falciparum malaria. In the present report we describe a highly sensitive methodology for malaria diagnosis using a nested PCR method based on amplification of the p126 P. falciparum gene detected by simple ethidium bromide staining. The P. falciparum Palo Alto strain (culture samples) was serially diluted in blood from an uninfected donor to a final level of parasitemia corresponding to 10(-8)% and was processed for PCR amplification. In each of these dilutions a parasitological examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained from 51 malarious patients with positive thick blood smears (TBS) who were living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ranging from only 16 to 20,200 parasites/microliter for P. falciparum disease and from 114 to 11,000 parasites/microliter for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/microliter without the use of radioactive material. The use of a 1-ml sample volume and the organic DNA extraction method should be suitable in blood banks and for the evaluation of patients during and after drug treatment.

Antibody responses to Plasmodium falciparum sporozoite-, liver- and blood-stage synthetic peptides in migrant and autochthonous populations in malaria endemic areas.

This study evaluates the differences in host immune responses to defined plasmodial antigens in four geographically different regions in which malaria is endemic. Sera from 527 individuals were tested for the presence of antibodies specific for three types of plasmodial antigen: liver-stage antigen (LSA-1), blood-stage antigen (SPF 70) and circumsporozoite (CS) antigen (NANP)4. The individuals taking part in the study comprised: patients with transfusional malaria due to Plasmodium falciparum or P. vivax; non-immune migrants residing in an endemic area in Rondônia; Amazonian Indians from the states of Pará (Xingu PA) and Mato Grosso (Xingu MT); people living in a hyperendemic area in Africa (Burkina-Faso); and controls that had never been to a malaria endemic area. None of the transfusional sera displayed antibodies against sporozoite or to liver stage antigen, although 80% of the P. falciparum transfusional malaria sera contained IgG antibodies against the blood-stage peptide. A low percentage of Indians from Xingu PA and of non-immune migrants displayed antibodies against liver-stage (27% and 17%) and sporozoite (11% or d 12%) peptides, although a greater frequency of antibodies against blood-stage peptide (50% and 49%) was observed in both cases. Indians from Xingu MT exhibited a greater frequency of antibodies against liver, sporozoite and blood-stage peptides (45%, 50% and 58%). Only hyperimmune African individuals exhibited higher percentages of antibodies against liver- (64%) and blood-stage antigens (87%), contrasting with a low frequency of antibodies against the CS repeat (33%). Taken together, the present data confirm that Rondonian migrants and Indians from Xingu PA constitute populations with limited exposure and immunity to P. falciparum malaria infection and conversely, Xingu MT Indians and Africans have been more exposed to malaria infection. In conclusion this study indicates that the immune response to these malaria parasite peptides can be used to assess malaria transmission in epidemiological surveys.

Malaria diagnosis: identification of an anti-40-kDa polypeptide antibody response associated with active or recent infection and study of the IgG/IgM ratio of antibodies to blood-stage Plasmodium falciparum antigens.

The need for an alternative methodology to assess disease activity in the case of malaria led us to evaluate the usefulness of studying the humoral immune response to establish the diagnosis of past or recent malaria. For this purpose, we analyzed sera from 439 individuals living in endemic areas of the Amazon region (Ariquemes, Rondonia). Individuals were classified according to the number and the date of past crises. The enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the IgG/IgM ratio so as to discriminate acute or recent malaria from past infections against crude and defined (SPF70) Plasmodium falciparum asexual blood-stage antigens. We also analyzed the humoral immune response against components presented in crude P. falciparum antigen by the immunoblot technique. Use of the IgG/IgM ratio values did not allow us to differentiate acute from past infections. However, when we analyzed the humoral immune response to parasite components, we were capable of identifying a polypeptide with a molecular weight ranging up to 40 kDa, which was recognized by all parasitized polyinfected individuals studied but not by individuals with negative thick blood smears. In view of these data, we conclude that the 40-kDa polypeptide may represent a powerful tool in the diagnosis of acute malaria, mainly for screening blood donors in endemic areas.

Tumor necrosis factor alpha interferon gamma and macrophage stimulating factor in relation to the Severity of Plasmodium falciparum malaria in the Brazilian Amazon.

We compared the tumor necrosis factor (TNF-alpha), interferon gamma (IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) serum levels in 87 patients with malaria from the Brazilian Amazon. They included asymptomatic infected individuals and symptomatic patients with mild disease or severe malaria with or without cerebral involvement. As controls we examined individuals living in endemic areas without past history of malaria. The TNF-alpha serum levels were increased in patients with malaria and progressively decreased in those with severe disease 7 days after specific treatment. We found correlation between parasitaemia, TNF-alpha levels and severity of the disease. The correlation between high TNF-alpha levels and severe malaria was independent of cerebral involvement. The increase in both IFN-gamma and GM-CSF levels among malarious patients was not related to the degree of severity or mortality. IFN-gamma concentration, however, was associated with high parasitaemia at admission.

Conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, in field isolates and distinct species of Plasmodium.

The extent of structural conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, recently characterized in the T9/96 clone, has been analyzed using the polymerase chain reaction. Results from Ivory Coast and Thai clones, field isolates originating from Brazil and Kenya and laboratory-maintained strains strongly suggest that this gene has a highly conserved structure throughout this species. This structure includes a complex repetitive central domain consisting of a mosaic region followed by tandem 45-amino acid-encoding (Rp45) and 10-amino acid-encoding (Rp10) repeat regions. Limited size variation in this domain appeared to result from highly localized duplication events in the Rp45 and Rp10 regions. No size variation was observed in the 5' and 3' coding non-repetitive regions, but minor size polymorphism was found in the single intron at the 5' end of the gene. No evidence was found of distinct families of polymorphic types, as has been observed with the blood-stage MSA-1, MSA-2 and S-antigens. The sequence of the STARP homologue in the phylogenetically close chimpanzee parasite, Plasmodium reichenowi, has also been elucidated and reveals high sequence conservation, although interesting differences were detected in the composition of the Rp10 region, known in P. falciparum to contain B- and T-cell epitopes. Finally, DNA hybridization reveals the presence in rodent malaria species of sequences containing homology to the STARP non-repetitive (though not the repetitive) regions, which would suggest that a similar, conserved gene may exist in these species.

Isolation and antigenicity of a 45-kilodalton Paracoccidioides brasiliensis immunodominant antigen.

In the present study, we analyzed human antibody responses to Paracoccidioides brasiliensis cellular antigens by the immunoblot technique to identify specific cellular components and to investigate the existence of antigen profile differences among serological responses of paracoccidioidomycosis (PCM) patients. Among the 64 PCM serum samples analyzed, a relatively homogeneous immunoglobulin G response to P. brasiliensis antigens was observed. The polypeptide with a mass of 45 kDa was the most clinically important, since antibody to this antigen was detectable in 90.6% of PCM patients studied and the six individuals who did not produce antibody were either at the end of treatment or in the posttherapy period and had shown clinical recovery. These facts suggested that the presence of this antibody may be an indicator of active disease. The 45-kDa antigen was also the most specific antigen of the PCM humoral immune response, since it reacted with only 2 of 79 (2.5%) heterologous serum samples tested: 1 histoplasmosis case and 1 tuberculosis case. This polypeptide was isolated from gels by electroelution and, when tested by an immunoradiometric assay and immunoblotting, maintained its reactivity with PCM sera and also with anti-P. brasiliensis polyclonal antibodies raised in rabbits at the same sensitivity levels as those obtained in immunoblotting with a crude antigen. Since in our assays the 45-kDa polypeptide was the major P. brasiliensis antigen and seemed to be specific for PCM, its use in alternative diagnostic methods is promising, especially in patients suspected of having the juvenile clinical form of PCM often associated with negative double-immunodiffusion results.

[Severe tricuspid valve insufficiency due to papillary anterior muscle infarction of the right ventricle secondary to neonatal hypoxia]. / Insuficiência tricúspide severa por infarto do músculo papilar anterior do ventrículo direito secundário à hipóxia neonatal.

A fatal case of a stressed newborn baby who developed tricuspid insufficiency due to an anterior papillary muscle infarction of the right ventricle, related to perinatal anoxia is reported. The baby needed resuscitation management and had a systolic murmur soon after birth. The echocardiographic examination showed a flail anterior leaflet of the tricuspid valve due to an avulsed anterior papillary muscle. There was rupture of chordae tendineae between anterior and posterior leaflet. Necrosis and calcium deposit were found in the area. The conclusion was that the anoxia in perinatal period, in most cases, causes transient lesions, but in few cases might be severe and fatal. It is important a complete cardiac evaluation in every case of prolonged perinatal stress.

Modern immunological approaches to assess malaria transmission and immunity and to diagnose plasmodial infection.

The present paper reviews our recent data concerning the use of immunological methods employing monoclonal antibodies and synthetic peptides to study malaria transmission and immunity and to diagnose plasmodial infection. As concerns malaria transmission, we studied the main vectors of human malaria and the plasmodial species transmitted in endemic areas of Rondônia state, Brazil. The natural infection of anopheline was evaluated by immunoradiometric assay (IRMA) using monoclonal antibodies to an immunodominant sporozoite surface antigen (CS protein) demonstrated to be species specific. Our results showed that among six species of Anopheles found infected, An. darlingi was the main vector transmitting Plasmodium falciparum and P. vivax malaria in the immediate vicinity of houses. In order to assess the level of anti-CS antibodies we studied, by IRMA using the synthetic peptide corresponding to the repetitive epitope of the sporozoite CS protein, sera of individuals living in the same areas where the entomological survey has been performed. In this assay the prevalence of anti-CS antibodies was very low and did not reflect the malaria transmission rate in the studied areas. In relation to malaria diagnosis, a monoclonal antibody specific to an epitope of a 50 kDa exoantigen, the major component of supernatant collected at the time of schizont rupture, was used as a probe for the detection of P. falciparum antigens. This assay seemed to be more sensitive than parasitological examination for malaria diagnosis since it was able to detect plasmodial antigens in both symptomatic and asymptomatic individuals with negative thick blood smear at different intervals after a last parasitologically confirmed attack of malaria.

Modern immunological approaches to assess malaria transmission and immunity and to diagnose plasmodial infection

The present paper reviews our recent data concerning the use of immunological methods employing monoclonal antibodies and synthetic peptides to study malaria transmission and immunity and to diagnose plasmodial infection. As concerns malaria transmission, we studied the main vectors of human malaria and the plasmodial species transmitted in endemic areas of Rondônia state, Brazil. The natural infection on anopheline was evaluated by immunoradiometric assay (IRMA) using monoclonal antibodies to an immunodominant sporozoite surface antigen (CS protein) demonstrated to be species specific. Our results showed that among six species of Anopheles found infected, An. darlingi was the main vector transmitting Plasmodium falciparum and P. vivax malaria in the immediate vicinity of houses. In order to assess the level of anti-CS antibodies we studied, by IRMA using the synthetic peptide corresponding to the repetitive epitope of the sporozoite CS protein, sera of individuals living in the same areas where the entomological survey has been performed. In this assay the prevalence of anti-CS antibodies was very low and did not reflect the malaria transmission rate in the studied areas. In relation to malaria diagnosis, a monoclonal antibody specific to an epitope of a 50 kDa exoantigen, the major component of supernatant collected at the time of schizont rupture, was used as a probe for the detection of P. falciparum antigens. This assay seemed to be more sensitive than parasitological examination for malaria diagnosis since it was able to detect plasmodial antigens in both symptomatic and asymtomatic individuals with negative thick blood smear at different intervals after a last parasitologically confirmed confirmed attack of malaria

Sensitive immunoradiometric assay for the detection of Paracoccidioides brasiliensis antigens in human sera.

In the present study we report the standardization of an immunoradiometric assay (IRMA) for detection of Paracoccidioides brasiliensis circulating antigens that could be useful in the diagnosis and prognosis of paracoccidioidomycosis. For this purpose we studied the reactivities of P. brasiliensis and other mycotic antigens with rabbit polyclonal anti-P. brasiliensis antibodies (immunoglobulin G) in order to evaluate the sensitivity and specificity of an IRMA for detecting P. brasiliensis antigens. The results were compared with those obtained by the double immunodiffusion test, the standard technique for the serodiagnosis of paracoccidioidomycosis. By using the immunoglobulin G fraction of rabbit antisera (900 ng per well), it was possible to detect up to 3.6 ng (0.12 micrograms/ml) of cellular antigen and 360 ng (12 micrograms/ml) of metabolic antigen in contrast to the double immunodiffusion test that could detect only 12 micrograms (1.2 mg/ml) of both antigens. IRMA was shown to be feasible and very sensitive and may therefore help, together with clinical data, in establishing early diagnosis and assessing disease activity. It could also allow the study of relationships between P. brasiliensis circulating antigens and host defense mechanisms during the disease.

Study of antibodies in paracoccidioidomycosis: follow-up of patients during and after treatment.

The relevance of the humoral response in the prognosis of paracoccidioidomycosis was assessed by measuring the serological responses of individual patients to Paracoccidioides brasiliensis by double immunodiffusion (DID). Sixty-six patients with paracoccidiodomycosis were studied. Sera from 31 individuals were tested before and during treatment with sulfonamide (Group I). Sera from a further 35 individuals were tested after completion of a 2-year course of treatment (Group II). In Group I, clinical improvement was associated with a decrease in antibody titer in all patients. The only patient in this group who had a clinical relapse during specific treatment presented with a 4-fold increase in antibody titer immediately before relapse. In Group II, nine patients remained antibody positive at follow-up (61.9 +/- 40.0 months), despite their good physical health, indicating that the detection of antibodies to P. brasiliensis by the DID test does not necessarily indicate active disease. These data suggest that changes in antibody titers to P. brasiliensis detected by DID may be useful indicators of the extent of active disease. Measurement of antibody titers may be valuable for determining the prognosis of the infection and for deciding on a suitable treatment protocol.

The possible role of circulating immune complexes in the deficiency of cell-mediated immunity in paracoccidioidomycosis.

1. The nature and extent of immune abnormalities was studied in 28 untreated patients with a chronic moderate form of paracoccidioidomycosis (PCM). 2. The patients presented hyporeactivity to skin tests, diminished lymphocyte transformation by mitogens such as phytohemagglutinin-P and concanavalin A and by Paracoccidioides brasiliensis protein antigen. They also presented peripheral blood leukocytosis but normal absolute numbers of T-cell and T-cell subsets. 3. The patients had increased serum levels of C3d, as well as high levels of circulating immune complexes (CIC) detected by C1q-binding and protein A-binding assays. 4. There was a significant negative correlation between lymphocyte transformation by mitogens and CIC levels which suggested that CIC may be involved in the genesis of the depressed cell-mediated immunity in PCM patients.

Aspergillus fumigatus fungus ball in hospitalized patients with chronic pulmonary disease. Usefulness of double immunodiffusion test as a screening procedure.

Double immunodiffusion (DID) was used as a screening test for the diagnosis of aspergillosis. Three hundred and fifty patients were tested, all of them referred from a specialized chest disease hospital and without a definitive etiological diagnosis. When DID was positive additional information such as clinical history and radiographic findings were requested and also surgical specimens were obtained whenever possible. Specific precipitin bands for Aspergillus fumigatus antigen were found in 29 (8.3%) of 350 patients sera. Nineteen (65.5%) of the 29 patients with positive serology were recognized as having a fungus ball by X-rays signs in 17 or by pathological examination in 2 or by both in 8 patients. This two-year prospective study has shown that pulmonary aspergillosis is a considerable problem among patients admitted to a Chest Diseases Hospital, especially in those with pulmonary cavities or bronchiectasis.