A nanochitosan-D-galactose formulation increases the accumulation of primaquine in the liver.

Primaquine is the mainstream antimalarial drug to prevent Plasmodium vivax relapses. However, this drug can induce hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency. Nanostructure formulations of primaquine loaded with D-galactose were used as a strategy to target the drug to the liver and decrease the hemolytic risks. Nanoemulsion (NE-Pq) and nanochitosan (NQ-Pq) formulations of primaquine diphosphate containing D-galactose were prepared and characterized by their physicochemistry properties. Pharmacokinetic and biodistribution studies were conducted using Swiss Webster mice. A single dose of 10 mg/kg of each nanoformulation or free primaquine solution was administered by gavage to the animals, which were killed at 0.5, 1, 2, 4, 8, and 24 hours. Blood samples and tissues were collected, processed, and analyzed by high-performance liquid chromatography. The nanoformulation showed sizes around 200 nm (NE-Pq) and 400 nm (NQ-Pq) and physicochemical stability for over 30 days. Free primaquine solution achieved higher primaquine Cmax in the liver than NE-Pq or NQ-Pq at 0.5 hours. However, the half-life and mean residence time (MRT) of primaquine in the liver were three times higher with the NQ-Pq formulation than with free primaquine, and the volume distribution was four times higher. Conversely, primaquine's half-life, MRT, and volume distribution in the plasma were lower for NQ-Pq than for free primaquine. NE-Pq, on the other hand, accumulated more in the lungs but not in the liver. Galactose-coated primaquine nanochitosan formulation showed increased drug targeting to the liver compared to free primaquine and may represent a promising strategy for a more efficient and safer radical cure for vivax malaria.

Pharmacotherapy follow-up: Role in active malaria surveillance in a travel medicine centre outside the transmission area in Brazil.

WHAT IS KNOWN AND OBJECTIVE: Malaria is a potentially severe disease, widespread in tropical and subtropical areas. Apart from parasite drug resistance, which receives the largest share of attention, several factors directly influence the response to antimalarial treatment such as incorrect doses, adverse drug events, lack of adherence to treatment, drug quality and drug-drug interactions. Pharmacotherapy follow-up can be used to monitor and improve the effectiveness of treatment, prevent drug-related problems and ensure patient safety. The aim of this study was to describe the results of the implementation of pharmacotherapy follow-up of patients with malaria seen at a reference centre for malaria diagnosis and treatment (CPD-Mal) located in the city of Rio de Janeiro, an area without malaria transmission. METHODS: A descriptive study was conducted from January 2009 to September 2013 at the Instituto Nacional de Infectologia Evandro Chagas (INI) of the Fundação Oswaldo Cruz (Fiocruz). All malaria patients enrolled in the study were treated according to the Brazilian Malaria Therapy Guidelines. Data collected during pharmacotherapy follow-up were recorded in a standardized form. The variables included were age, gender, comorbidities, antimalarials and concomitant medications used, adverse drug reactions (ADR), clinical and parasitological cure times, and treatment outcomes classified as success, recurrence (recrudescence or relapse); and lost to follow-up. The ADR were classified by severity (DAIDS-NIH), organ system affected (WHO-ART) and likelihood to be caused by drugs (Naranjo scale). RESULTS AND DISCUSSION: One hundred thirteen cases of malaria were included. Patients were aged between 13 and 66 years and the majority of them (75.2%) were male. Ninety-four ADR were observed, most classified as mild (85.1%), related to disorders of the gastrointestinal system (63.8%), such as nausea and vomiting, and assessed as "possibly" caused by the antimalarial drugs (91.5%). The majority of clinical (90.9%) and parasitological (87.1%) cure occurred less than 72 hours after treatment initiation. Pharmacotherapy follow-up of malaria treatment by surveillance activities is therefore important regarding information about treatment outcomes as well as patient safety, resulting in better patient care and reducing the chance of relapses. The results underscore its use as a tool for monitoring adherence and drug resistance outside an endemic area. WHAT IS NEW AND CONCLUSION: Pharmacotherapy follow-up should be considered a useful malaria surveillance tool that can be developed by reference centres for comprehensive health care assistance and monitoring of therapeutic resistance.

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.

DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus, two non-human primate models for malaria.

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFß2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most--except IL3 and IL4--showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024, and FJ89029.

[Reflections on Three Episodes of Louis Pasteur's Life as Seen in the William Dieterle's Movie (1936)]. / Réflexions sur trois épisodes de la vie de Louis Pasteur vus au travers du film de William Dieterle (1936).

Despite the criticism and reservations made about him still nowadays, Louis Pasteur may be considered one of the most important scientists of the last two centuries in public health, even if the work of the numerous scientists who preceded him have largely contributed to the successes he obtained without following too much to the rules of deontology and ethics currently in force in the world of research and medicine. He has definitively put down, by his experiments, the "theory of spontaneous generation" in force since antiquity, validated that of "germs or microbes", enacted the first rules of asepsis, while inspiring those of the antisepsis applied by Joseph Lister, and developed a certain number of vaccinations in veterinary and human medicine, including the anti-rabies, the one which made him famous all over the world. All this was not done without difficulty and Pasteur encountered for a large part of his life the misunderstanding of his contemporaries and the hostility of the medical world to which he did not belong. The authors comment in this text the movie The Story of Louis Pasteur by William Dieterle, filmed in 1936, based on the knowledge acquired since that date and doing the part of the real and the fiction.

Malgré les critiques et les réserves dont il est l'objet aujourd'hui, Louis Pasteur peut être considéré comme l'un des scientifiques les plus importants de ces deux derniers siècles en matière de santé publique, même si les apports des nombreux hommes de science qui l'ont précédé ont largement contribué aux succès qu'il obtint sans trop se soucier des règles de déontologie et d'éthique actuellement en vigueur dans le monde de la recherche et de la médecine. Il a mis définitivement à bas, par ses expériences, la « théorie de la génération spontanée ¼ en vigueur depuis l'Antiquité, validé celle « des germes ou microbes ¼, édicté les premières règles de l'asepsie tout en inspirant celles de l'antisepsie appliquée par Joseph Lister et mit au point un certain nombre de vaccinations en médecine vétérinaire puis humaine, notamment celle contre la rage, ce qui le rendit célèbre dans le monde entier. Tout cela ne s'est pas fait sans difficulté, et Pasteur s'est heurté pendant une grande partie de sa vie à l'incompréhension de ses contemporains et à l'hostilité du monde médical auquel il n'appartenait pas. Les auteurs commentent dans ce texte le film L'Histoire de Louis Pasteur de William Dieterle, tourné en 1936, en s'appuyant sur les connaissances acquises depuis cette date et en faisant la part du réel et de l'imaginé.

[Plasmodium vivax malaria in the Brazilian Amazon: some aspects of its epidemiology, clinical spectrum and naturally induced immune responses]. / Paludisme dû a Plasmodium vivax en Amazonie brésilienne: quelques aspects de son epidémiologie, de ses manifestations cliniques et des réactions immunitaires naturellement acquises.

Malaria was a nationwide problem in Brazil in the 1940's. However during the late fifties a national and successful campaign gained strength in the country decreasing malaria to its lowest level by 1960, when only 36,9 thousand cases were registered. Although the Malaria Eradication Program of the Ministry of Health in Brazil succeeded by the late 60's in freeing the majority of the country from malaria transmission, it was unable to contain the rapid spread of the disease in the Amazon Basin. In the 1970's the Amazon region witnessed a huge transformation. Colonization programs sponsored by the government, mining exploration, massive migration and the construction of roads and dams brought a new reality for which the area was not prepared. The last data available show that in 2007, the Amazon registered around 450 thousand cases, 99.9% of the national cases. P. vivax has been reported as representing around 80% of all malaria cases. P. vivax is thought to cause little mortality but like P. falciparum, P. vivax accounts for vast amounts of morbidity and for huge burdens on the prosperity of endemic communities. However, in the last few years a pattern of unusual clinical complications with fatal cases associated with it have been reported in Brazil and is a matter of tremendous concern in the Brazilian community of malariologists. In addition, the emergence of P. vivax strains resistant to chloroquine and primaquine in some reports needs to be further investigated. In contrast, asymptomatic infections by P. falciparum and P. vivax were detected in epidemiological studies in the states of Rondonia and Amazonas. Seropidemiological studies investigating the type of immune responses elicited in naturally exposed populations to several malaria vaccine candidates in Brazilian populations have also been providing important information on whether immune responses specific to those antigens are generated in natural infections and their immunogenic potential as vaccine candidates. In fact ultimate test of a malaria vaccine will be determined in field trials under natural conditions of parasite exposure.

Natural malaria infections in anophelines in Rondonia State, Brazilian Amazon.

The use of an Immunoassay for the detection of Plasmodium falciparum and P. vivax circumsporozoite (CS) antigens in anophelines has recently incriminated other malaria vectors besides Anopheles darlingi in the Brazilian Amazon. In this study we analyzed 12,336 field-collected anophelines from endemic areas in Rondonia for plasmodial infection. Sixty-one specimens from 6 species were positive: 47 An. darlingi, 5 An. triannulatus, 4 An. albitarsis, 2 An. braziliensis, 2 An. strodei, and 1 An. oswaldoi. As concerns the species, 41 anopheles harbored P. falciparum and 20 were infected with P. vivax. An. darlingi was the most important local vector, as it was the one most frequently found infected and the only one clearly related to areas where malaria transmission was being recorded.

Immune response and lack of immune response to Plasmodium falciparum P126 antigen and its amino-terminal repeat in malaria-infected humans.

A parasitophorous vacuole protein of Plasmodium falciparum, p126, is a potential candidate for a malaria vaccine. Its N-terminal region, composed of six repeats of eight amino acids, appears to be involved in the induction of protective immunity against P. falciparum challenge in monkeys. This study evaluated the immune response to p126 and to its N-terminal region (Nt47) in patients (n = 45) living in a malaria-endemic area of Brazil (Colina, Porto Velho, Rondonia). Cellular proliferative responses against Nt47 were low and infrequent. The study of the humoral immune response demonstrated that 95% of the patients had detectable anti-p126 antibodies and 77% had anti-Nt47 antibodies. Analysis of the antibody isotypes specific for Nt47 revealed that all four IgG subclasses were present and individuals with higher levels of anti-Nt47 cytophilic IgG antibody (IgG1 + IgG3/IgG2 + IgG4) had significantly lower parasitemia levels, suggesting that antibodies to the N-terminal region of the p126 protein may contribute to acquisition of immunity to P. falciparum malaria.

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.

Abnormal anti-single stranded (ss) DNA activity in sera from Plasmodium falciparum infected individuals.

Sera from 32 subjects with Plasmodium falciparum parasitaemia were screened for the presence of antibodies to native-double stranded (ds) DNA and to heat-denaturated-single stranded (ss) DNA by a Farr DNA binding radioimmunoassay. In addition anti-dsDNA antibodies were also studied by indirect immunofluorescence using Crithidia luciliae and rat liver sections as substrates. Immunoglobulin (G, A, M) levels and plasmodial antibodies titres (PA) were concomitantly evaluated. The anti-ssDNA activity was higher in malarious individuals with high IgM levels than in normal or malarious individuals with normal IgM levels. This activity was higher during the acute stage of infection than after recovery. A positive and significant relationship was found between the anti-ssDNA activity and the IgM levels but not with IgG, IgA or PA titres. Speckled antinuclear antibodies (ANA) were also observed in sera from 43.8% of the individuals and the mean ssDNA activity was higher in these ANA positive patients. Conversely anti-dsDNA antibodies could not be detected by any of the tests performed. This preferential production of anti-ssDNA Ab and not anti-dsDNA Ab is additional evidence that the autoantibodies observed in malaria infection are not a consequence of a generalized and non-specific polyclonal activation.

Autoimmunity and malaria: what are they doing together?

A common feature of autoimmunity is the presence of autoantibodies (AAb). Two types of AAb have been described: the 'pathogenic' AAb, associated with autoimmune diseases (AID), and the so-called 'natural' AAb. The latter are present in all normal individuals and have been postulated to play a major role as a first defensive barrier of the organism. Both the 'pathogenic' and the 'natural' AAb can be detected at higher frequencies among individuals exposed to viral, bacterial and parasitic infections. The malaria associated AAb do not seem to result from a generalised polyclonal B-cell activation (PBA), have specificities that may differ according to the degree of clinical immunity and do not seem to be pathogenic. Malaria may offer a protective effect against AID, by diminishing its severity or by either preventing or retarding its expression. AAb could also participate in the immune protection against malaria, and this could happen in several ways: (i) AAb directed to modified Ag expressed on the red blood cell (RBC) membrane during parasitisation and (ii) AAb reactive with crypto- or neo-Ag revealed on both normal and infected RBC membranes, by destroying infected, and also normal, erythrocytes; (iii) anti-idiotype AAb specific of the binding site of anti-merozoite Ab, which would mimic the parasite ligand for the RBC receptor, by competing with parasites and blocking RBC invasion; (iv) AAb cross-reactive with parasite material - such as nuclear or cytoskeleton Ag - having a direct parasiticide activity; (v) the natural AAb network, through its 'anti-bacterial first defense barrier'; and finally (vi) anti-phospholipid (PL) AAb, by neutralizing the pathogenic properties of parasite-derived PL. Finally, in view of currently available knowledge, it is concluded that, since AAb are not always pathogenic, the price for an 'autoimmunity-mediated' protection in malaria would not necessarily be immunopathology and clinical autoimmunity, and a protective role of AAb could be exerted with no danger to the host.

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.

Clinical and laboratory findings of disseminated Mycobacterium avium complex infection (DMAC) in a pair matched case-control study.

A pair matched case/control study was conducted from January 1991 to 30 June 1992 in order to define clinical and laboratory findings associated with DMAC infection in AIDS patients. Since DMAC infection is usually associated with advanced immunodeficiency, and therefore also with other opportunistic illnesses, in addition to the number of CD4+ lymphocytes, cases and controls were matched using the following criteria: date of AIDS diagnosis and antiretroviral therapy, number and severity of associated opportunistic infections and, whenever possible, type of Pneumocystis carinii prophylaxis, age and gender, in this order of relevance. Cases (defined as patients presenting at least one positive culture for MAC at a normally sterile site) and controls presented CD4+ lymphocyte counts below 50 cel/mm3. A significantly higher prevalence of general, digestive and respiratory signs, increased LDH levels, low hemoglobin levels and CD4+ cell counts were recorded for cases when compared to controls. Increases in gammaGT and alkaline phosphatase levels seen in cases were also recorded for controls. In conclusion, the strategy we used for selecting controls allowed us to detect laboratory findings associated to DMAC infection not found in other advanced immunosupressed AIDS patients without DMAC.

[Vaccines: a public affair?] [corrected]. / Vacinas: negócio de Estado?

In spite of the expectation that the development of vaccines would help in the control of some of the main transmissible diseases in Brazil, which are responsible for a large share of endemic illness in the country, efficient and safe vaccines against no less serious contagious diseases are available, yet underutilized. The reason is simple: some vaccines are not included in the National Immunization Program (PNI) and are this not the object of government campaigns. Despite the competence with which the National Health Foundation has acted-through the PNI-in reducing prevalence of diseases such as diphteria, whooping cough, measles, and polio, lack of information and prejudice make several vaccines inaccessible to a major share of the Brazilian population. Since the government-through its vaccination campaigns-exerts the most important influence on public opinion in this area, a vaccine procedure which is not the object of such campaigns will obviously not become part of popular culture. The result of this lack of information is that the population fails to utilize vaccines against such diseases as rubella, mumps, and hepatitis B or against Haemophilus influenza type b and pneumococcus infections. We therefore argue for information on the existence and availability of these vaccines, so that not only the part of the population that has access to private medicine can benefit, but mainly so that pressure can be brought to bear on the government for all public health care services to supply them to the neediest portion of the population. We suggest that strategies for the socialization of immunoprophylatics should be defined with participation by all segments of society, incorporating measures such as the gradual expansion of the National Immunization Program, coverage of vaccine costs by health insurance policies, and granting of fiscal incentives to companies that vaccinate their employees and families. Finally, we propose a struggle against the prejudice surrounding participation by private initiative in collective health actions, as well as defense of the interaction between private and public sectors in all aspects of health throughout Brazil.

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.

Naturally acquired humoral and cellular immune responses to Plasmodium vivax merozoite surface protein 9 in Northwestern Amazon individuals.

Antibody and T-cell reactivities to Plasmodium vivax merozoite surface protein 9 (PvMSP9) were evaluated in a cross-sectional study of individuals naturally exposed to malaria infections living in Ribeirinha, a native riverine community and in Colina, a transmigrant community, Rondonia, Brazil. The antibody responses to PvMSP9-RIRIIand PvMSP9-Nt domains in Ribeirinha were higher compared with Colina and correlated with age and time of malaria exposure. IgG2 was most prevalent for PvMSP9-RII in both communities, and IgG1 was the predominant isotype for PvMSP9-Nt and PvMSP9-RIRII in Ribeirinha. IFN-gamma and IL-4 predominated in Ribeirinha, while IFN-gamma predominated in Colina. Variation in exposure to P. vivax likely accounts for the differences observed in cytokine and antibody levels between the two populations studied.

Is there a role for autoimmunity in immune protection against malaria?

Much remains to be known about the mechanisms involved in protective immunity against malaria and the way it is acquired. This is probably the reason why, in spite of so much progress, it has not yet been possible to develop an anti-malaria vaccine able to induce parasite specific antibodies (Ab) and/or T-cells. It has been considered in the early 80s that the induction of efficient protection against the blood stage forms of Plasmodium falciparum would not be possible without simultaneously eliciting an autoimmune (AI) response against erythrocytes, even at the price of inducing an AI pathology. Despite the description of the reciprocal relationship, i.e. the protective effect of malaria on the development of AI diseases _ demonstrated since 1970 _ no effort has been made to verify the possible involvement of the AI response in protection against 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.

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.