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Background: Although blood transfusion is an essential therapeutic procedure, it can present risks, including transmitting infectious diseases, such as malaria. In Acre, the thick blood smear microscopic examination (TBS) is used to screen infected malaria blood donors. However, TBS has low sensitivity for detecting Plasmodium in situations of low parasitaemia, such as those presented by asymptomatic clinically healthy individuals., Objectives: To investigate the pertinence of using polymerase chain reaction (PCR) to detect malarial infection for screening blood donors in Cruzeiro do Sul, Acre, an endemic high-risk malaria area in the Legal Amazon., Methods: A cross-sectional study was conducted among individuals eligible and ineligible to be blood donors, according to clinical and epidemiological criteria. Besides the mandatory screening of HCV, HBV, and HIV tests, malaria PCR and TBS were also carried out on all blood donor candidates who attended the Cruzeiro do Sul Blood Centre from July to September 2022., Findings: Of the 230 participants, 209 (91%) were eligible for blood donation by clinical-epidemiological screening. Surprisingly, no blood donor candidate reported a history of malaria. All TBS microscopic tests were negative at the time of recruitment. However, samples from four blood donor candidates (two eligible by clinical and epidemiological malaria criteria and two ineligible by hypertension and recent tattoo) were positive by Plasmodium and P. vivax molecular tests., Main Conclusions: Malaria molecular techniques for screening blood donors should be introduced in the Brazilian Blood Centres to maximise recipient safety. Furthermore, selecting zero-risk donors could pave the way to build a transmissible malaria-free environment in the blood bank context in the near future.

Plasmodium vivax causes the largest malaria burden in Brazil, and chloroquine resistance poses a challenge to eliminating malaria by 2035. Illegal mining in the Roraima Yanomami Indigenous territory can lead to the introduction of resistant parasites. This study aimed to investigate mutations in the pvcrt-o and pvmdr-1 genes to determine their potential as predictors of P. vivax chloroquine-resistant phenotypes. Samples were collected in two health centers of Boa Vista. A questionnaire was completed, and blood was drawn from each patient. Then, DNA extraction, PCR, amplicon purification, and DNA sequencing were performed. After alignment with the Sal-1, the amplified fragment was analyzed. Patients infected with the mutant parasites were queried in the Surveillance Information System. Among the patients, 98% (157/164) of participants were from illegal mining areas. The pvcrt-o was sequenced in 151 samples, and the K10 insertion was identified in 13% of them. The pvmdr1 was sequenced in 80 samples, and the M YF haplotype (958 M ) was detected in 92% of them and the TYF was detected in 8%, while the M Y L was absent. No cases of recrudescence, hospitalization, or death were found. Mutations in the pvcrt-o and pvmdr-1 genes have no potential to predict chloroquine resistance in P. vivax .

Malaria is a public health problem and the cases diagnosed in the capital of Roraima, Brazil, show potential to characterize the burden of the disease in the state. This study aimed to describe the epidemiological, clinical, and laboratory aspects of malaria cases diagnosed in Boa Vista. For this purpose, a descriptive, cross-sectional study was conducted in two health units in the city, with individuals diagnosed and who agreed to respond the questionnaire. Of the total of 206 participants, characterized as men, mixed-race, and young, 96% (198) reported participating in illegal mining activity. Among the group of miners, 66% (131) came from other states of Brazil or other countries. The mines were mainly located in the Yanomami territory in Roraima. Plasmodium vivax infection occurred in 74% (153) of participants. In the miner's group, hospitalizations for severe malaria, previous malaria attacks, and delays in treatment after the onset of symptoms were reported. Although 73% (145) of miners reported knowing how malaria was transmitted, only 54% (107) used mosquito nets or repellents. The use of Artecom and chloroquine by miners is not for the complete treatment but only to relieve symptoms for returning to gold mines, highlighting the importance of molecular surveillance to antimalarial resistance. Indigenous peoples are considered vulnerable to malaria and miners promotes the increase of malaria in Roraima Indigenous Lands. Therefore, access to diagnosis and treatment in Indigenous areas invaded by miners is imperative to confront this disease that ravages Indigenous communities and threatens public health on a large scale to achieve the goal of eliminating malaria in the state.

Multidrug- and artemisinin-resistant (ART-R) Plasmodium falciparum (Pf) parasites represent a challenge for malaria elimination worldwide. Molecular monitoring in the Kelch domain region (pfk13) gene allows tracking mutations in parasite resistance to artemisinin. The increase in illegal miners in the Roraima Yanomami indigenous land (YIL) could favor ART-R parasites. Thus, this study aimed to investigate ART-R in patients from illegal gold mining areas in the YIL of Roraima, Brazil. A questionnaire was conducted, and blood was collected from 48 patients diagnosed with P. falciparum or mixed malaria ( Pf + P. vivax ). The DNA was extracted and the pfk13 gene was amplified by PCR. The amplicons were subjected to DNA-Sanger-sequencing and the entire amplified fragment was analyzed. Among the patients, 96% (46) were from illegal mining areas of the YIL. All parasite samples carried the wild-type genotypes/ART-sensitive phenotypes. These data reinforce the continued use of artemisinin-based combination therapies (ACTs) in Roraima, as well as the maintenance of systematic monitoring for early detection of parasite populations resistant to ART, mainly in regions with an intense flow of individuals from mining areas, such as the YIL. This is especially true when the achievement of falciparum malaria elimination in Brazil is planned and expected by 2030.

(1) Background: Malaria remains a significant global public health issue. Since parasites quickly became resistant to most of the available antimalarial drugs, treatment effectiveness must be constantly monitored. In Brazil, up to 10% of cases of vivax malaria resistant to chloroquine (CQ) have been registered. Unlike P. falciparum , there are no definitive molecular markers for the chemoresistance of P. vivax to CQ. This work aimed to investigate whether polymorphisms in the pvcrt-o and pvmdr1 genes could be used as markers for assessing its resistance to CQ. (2) Methods: A total of 130 samples from P. vivax malaria cases with no clinical and/or parasitological evidence of CQ resistance were studied through polymerase chain reaction for gene amplification followed by target DNA sequencing. (3) Results: In the pvcrt-o exons, the K10 insert was present in 14% of the isolates. Regarding pvmdr1 , T958 M and F1076 L haplotypes showed frequencies of 95% and 3%, respectively, while the SNP Y976 F was not detected. (4) Conclusions: Since K10- pvcrt-o and F1076 L /T958 M - pvmdr1 polymorphisms were detected in samples from patients who responded well to CQ treatment, it can be concluded that mutations in these genes do not seem to have a potential for association with the phenotype of CQ resistance.

(1) Background: Malaria is a public health problem worldwide. Despite global efforts to control it, antimalarial drug resistance remains a great challenge. In 2009, our team identified, for the first time in Brazil, chloroquine (CQ)-susceptible Plasmodium falciparum parasites in isolates from the Brazilian Amazon. The present study extends those observations to include survey samples from 2010 to 2018 from the Amazonas and Acre states for the purpose of tracking pfcrt molecular changes in P. falciparum parasites. (2) Objective: to investigate SNPs in the P. falciparum gene associated with chemoresistance to CQ ( pfcrt) . (3) Methods: Sixty-six P. falciparum samples from the Amazonas and Acre states were collected from 2010 to 2018 in patients diagnosed at the Reference Research Center for Treatment and Diagnosis of Malaria (CPD-Mal/Fiocruz), FMT-HVD and Acre Health Units. These samples were subjected to PCR and DNA Sanger sequencing to identify mutations in pfcrt (C72 S , M74 I , N75 E , and K76 T ). (4) Results: Of the 66 P. falciparum samples genotyped for pfcrt , 94% carried CQ-resistant genotypes and only 4 showed a CQ pfcrt sensitive-wild type genotype, i.e., 1 from Barcelos and 3 from Manaus. (5) Conclusion: CQ-resistant P. falciparum populations are fixed, and thus, CQ cannot be reintroduced in malaria falciparum therapy.

Background: Endemic malaria is present in all 15 municipalities of Roraima state, Brazilian Amazonia. Knowledge of epidemiological data of specific populations can guide health policies to formulate effective strategies for integrated control of health-disease care. This study aims to ascertain when, where and who fell ill with malaria in Roraima state from 2010 to 2020., Methods: This descriptive study was based on statistical secondary surveillance data through the analysis of relationships underlying numbers of cases, hospitalizations and deaths using the Malaria Epidemiological Surveillance Information System, Mortality Information System and Hospitalization Information System., Results: From 2010 to 2020, there were 138,504 autochthonous cases, 26,158 Venezuelan imported cases, 3765 hospitalizations, and 77 deaths from malaria reported in Roraima. Annual parasitic incidence and the number of hospitalizations showed impressive changes over the period, but without significantly correlating with number of deaths. The proportion of Plasmodium falciparum infections had significant shifts throughout this study. Malaria prevalence in indigenous and mining areas has been increasing since 2014., Conclusion: The presence of miners in indigenous areas is a reality that has been contributing to the increase of malaria cases in Roraima. The need to implement health policies that also meet this contingent is reinforced., (© 2022. The Author(s).)

Artemisinin (ART) is recommended as the first-line drug for P. falciparum infections combined with a long-acting partner drug. The emergence of P. falciparum resistance to ART (ARTR) is a concern for malaria. The most feared threat remains the spread of ARTR from Southeast Asia to Africa or the independent emergence of ARTR in Africa, where malaria accounts for 93% of all malaria cases and 94% of deaths. To avoid this worst-case scenario, surveillance of Pfkelch13 mutations is essential. We investigated mutations of Pfkelch13 in 78 P. falciparum samples from Huambo, Angola. Most of the parasites had a wild-type Pfkelch13 allele. We identified one synonymous mutation (R471 R ) in 10 isolates and one non-synonymous mutation (A578 S ) in two samples. No Pfkelch13 validated or candidate ARTR mutants were identified. The finding suggests that there is little polymorphism in Pfkelch13 in Huambo. Since cases of late response to ART in Africa and the emergence of ARTR mutations in Rwanda and Uganda have been reported, efforts should be made toward continuous molecular surveillance of ARTR. Our study has some limitations. Since we analyzed P. falciparum parasites from a single health facility, the study may not be representative of all Angolan endemic areas.

[This corrects the article DOI: 10.1371/journal.pone.0241426.].

Background: Plasmodium simium, a malaria parasite of non-human primates (NHP), was recently shown to cause zoonotic infections in humans in Brazil. We sequenced the P. simium genome to investigate its evolutionary history and to identify any genetic adaptions that may underlie the ability of this parasite to switch between host species., Results: Phylogenetic analyses based on whole genome sequences of P. simium from humans and NHPs reveals that P. simium is monophyletic within the broader diversity of South American Plasmodium vivax, suggesting P. simium first infected NHPs as a result of a host switch of P. vivax from humans. The P. simium isolates show the closest relationship to Mexican P. vivax isolates. Analysis of erythrocyte invasion genes reveals differences between P. vivax and P. simium, including large deletions in the Duffy-binding protein 1 (DBP1) and reticulocyte-binding protein 2a genes of P. simium. Analysis of P. simium isolated from NHPs and humans revealed a deletion of 38 amino acids in DBP1 present in all human-derived isolates, whereas NHP isolates were multi-allelic., Conclusions: Analysis of the P. simium genome confirmed a close phylogenetic relationship between P. simium and P. vivax, and suggests a very recent American origin for P. simium. The presence of the DBP1 deletion in all human-derived isolates tested suggests that this deletion, in combination with other genetic changes in P. simium, may facilitate the invasion of human red blood cells and may explain, at least in part, the basis of the recent zoonotic infections., (© 2021. The Author(s).)

Unforeseen Plasmodium infections in the Atlantic Forest of Brazilian Extra-Amazonian region could jeopardise malaria elimination. A human malaria case was registered in Três Forquilhas, in the Atlantic Forest biome of Rio Grande do Sul, after a 45 years' time-lapsed without any malaria autochthonous notification in this southern Brazilian state. This finding represents the expansion of the malaria distribution areas in Brazil and the southernmost human malaria case record in South America in this decade. The coexistence of the bromeliad-breeding vector Anopheles (Kerteszia) cruzii and non-human primates in the Atlantic Forest regularly visited by the patient claimed for the zoonotic origin of this infection. The reemergence of Atlantic Forest human malaria in Rio Grande do Sul was also discussed.

Plasmodium vivax merozoite surface proteins (PvMSP) 1 and 7 are considered vaccine targets. Genetic diversity knowledge is crucial to assess their potential as immunogens and to provide insights about population structure in different epidemiological contexts. Here, we investigate the variability of pvmsp-1 42 , pvmsp-7E, and pvmsp-7F genes in 227 samples from the Brazilian Amazon (BA) and Rio de Janeiro Atlantic Forest (AF). pvmsp-1 42 has 63 polymorphisms - 57 nonsynonymous - generating a nucleotide diversity of π = 0.009 in AF, and π = 0.018 in BA. In pvmsp-7E, 134 polymorphisms - 103 nonsynonymous - generate the nucleotide diversity of π = 0.027 in AF, and π = 0.042 in BA. The pvmsp-7F has only two SNPs - A610G and A1054T -, with nucleotide diversity of π = 0.0004 in AF, and π = 0.0007 in BA. The haplotype diversity of pvmsp-1 42 , pvmsp-7E, and pvmsp-7F genes is 0.997, 1.00, and 0.649, respectively. None of the pvmsp-1 42 or pvmsp-7E sequences are identical to the Salvador 1 strain's sequence. Conversely, most of pvmsp-7F sequences (94/48%) are identical to Sal-1. We evaluated eight B-cell epitopes in pvmsp-7E, four of them showed higher nucleotide diversity compared to pvmsp-7E's epitopes. Positive selection was detected in pvmsp-1 42 , pvmsp-7E central region, and pvmsp-7F with Tajima's D. In pvmsp-7E, the significant nucleotide and haplotype diversities with low genetic differentiation, could be indicative of balancing selection. The genetic differentiation of pvmsp-1 42 (0.315) and pvmsp-7F (0.354) genes between AF and BA regions is significant, which is not the case for pvmsp-7E (0.193). We conclude that pvmsp-1 42 and pvmsp-7E have great genetic diversity even in AF region, an enclosure area with deficient transmission levels of P. vivax zoonotic malaria. In both Brazilian regions, pvmsp-1 19 , pvmsp-7E, and pvmsp-7F are conserved, most likely due to their roles in parasite survival, and could be considered potential targets for a "blood-stage vaccine"., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Circumsporozoite protein (CSP) is the primary pre-erythrocytic vaccine target in Plasmodium species. Knowledge about their genetic diversity can help predict vaccine efficacy and the spread of novel parasite variants. Thus, we investigated pvcsp gene polymorphisms in 219 isolates (136 from Brazilian Amazon [BA], 71 from Rio de Janeiro Atlantic Forest [AF], and 12 from non-Brazilian countries [NB]). Forty-eight polymorphic sites were detected, 46 in the central repeat region (CR), and two in the C-terminal region. Also, the CR presents InDels and a variable number of repeats. All samples correspond to the VK210 variant, and 24 VK210 subtypes based on CR. Nucleotide diversity (π = 0.0135) generated a significant number of haplotypes (168) with low genetic differentiation between the Brazilian regions (Fst = 0.208). The haplotype network revealed similar distances among the BA and AF regions. The linkage disequilibrium indicates that recombination does not seem to be acting in diversity, reinforcing natural selection's role in accelerating adaptive evolution. The high diversity (low Fst) and polymorphism frequencies could be indicators of balancing selection. Although malaria in BA and AF have distinct vector species and different host immune pressures, consistent genetic signature was found in two regions. The immunodominant B-cell epitope mapped in the CR varies from seven to 19 repeats. The CR T-cell epitope is conserved only in 39 samples. Concerning to C-terminal region, the Th2R epitope presented nonsynonymous SNP only in 6% of Brazilian samples, and the Th3R epitope remained conserved in all studied regions. We conclude that, although the uneven distribution of alleles may jeopardize the deployment of vaccines directed to a specific variable locus, a unique vaccine formulation could protect populations in all Brazilian regions., Competing Interests: The authors have declared that no competing interests exist.

Background: Plasmodium vivax is the most widespread human malaria parasite outside Africa and is the predominant parasite in the Americas. Increasing reports of P. vivax disease severity, together with the emergence of drug-resistant strains, underscore the urgency of the development of vaccines against P. vivax. Polymorphisms on DBP-II-gene could act as an immune evasion mechanism and, consequently, limited the vaccine efficacy. This study aimed to investigate the pvdbp-II genetic diversity in two Brazilian regions with different epidemiological patterns: the unstable transmission area in the Atlantic Forest (AF) of Rio de Janeiro and; the fixed malaria-endemic area in Brazilian Amazon (BA)., Methods: 216 Brazilian P. vivax infected blood samples, diagnosed by microscopic examination and PCR, were investigated. The region flanking pvdbp-II was amplified by PCR and sequenced. Genetic polymorphisms of pvdbp-II were estimated based on the number of segregating sites and nucleotide and haplotype diversities; the degree of differentiation between-regions was evaluated applying Wright's statistics. Natural selection was calculated using the rate of nonsynonymous per synonymous substitutions with the Z-test, and the evolutionary distance was estimated based on the reconstructed tree., Results: 79 samples from AF and 137 from BA were successfully sequenced. The analyses showed 28 polymorphic sites distributed in 21 codons, with only 5% of the samples Salvador 1 type. The highest rates of polymorphic sites were found in B- and T cell epitopes. Unexpectedly, the nucleotide diversity in pvdbp-II was higher in AF (0.01) than in BA (0.008). Among the 28 SNPs detected, 18 are shared between P. vivax isolates from AF and BA regions, but 8 SNPs were exclusively detected in AF-I322S, K371N, E385Q, E385T, K386T, K411N, I419L and I419R-and 2 (N375D and I419M) arose exclusively in BA. These findings could suggest the potential of these geographical clusters as population-specific-signatures that may be useful to track the origin of infections. The sample size should be increased in order to confirm this possibility., Conclusions: The results highlight that the pvdbp-II polymorphisms are positively selected by host's immune pressure. The characterization of pvdbp-II polymorphisms might be useful for designing effective DBP-II-based vaccines.

Background: Although malaria cases have substantially decreased in Southeast Brazil, a significant increase in the number of Plasmodium vivax-like autochthonous human cases has been reported in remote areas of the Atlantic Forest in the past few decades in Rio de Janeiro (RJ) state, including an outbreak during 2015-2016. The singular clinical and epidemiological aspects in several human cases, and collectively with molecular and genetic data, revealed that they were due to the non-human primate (NHP) parasite Plasmodium simium; however, the understanding of the autochthonous malarial epidemiology in Southeast Brazil can only be acquired by assessing the circulation of NHP Plasmodium in the foci and determining its hosts., Methodology: A large sampling effort was carried out in the Atlantic forest of RJ and its bordering states (Minas Gerais, São Paulo, Espírito Santo) for collecting and examining free-living NHPs. Blood and/or viscera were analyzed for Plasmodium infections via molecular and microscopic techniques., Principal Findings: In total, 146 NHPs of six species, from 30 counties in four states, were tested, of which majority were collected from RJ. Howler monkeys (Alouatta clamitans) were the only species found infected. In RJ, 26% of these monkeys tested positive, of which 17% were found to be infected with P. simium. Importantly, specific single nucleotide polymorphisms-the only available genetic markers that differentiate P. simium from P. vivax-were detected in all P. simium infected A. clamitans despite their geographical origin of malarial foci. Interestingly, 71% of P. simium infected NHPs were from the coastal slope of a mountain chain (Serra do Mar), where majority of the human cases were found. Plasmodium brasilianum/malariae was initially detected in 14% and 25% free-living howler monkeys in RJ and in the Espírito Santo (ES) state, respectively. Moreover, the malarial pigment was detected in the spleen fragments of 50% of a subsample comprising dead howler monkeys in both RJ and ES. All NHPs were negative for Plasmodium falciparum., Conclusions/significance: Our data indicate that howler monkeys act as the main reservoir for the Atlantic forest human malarial parasites in RJ and other sites in Southeast Brazil and reinforce its zoonotic characteristics., Competing Interests: The authors have declared that no competing interests exist.

Background and Objective: Brazil is responsible for a large number of Plasmodium vivax cases in America. Given the emergence of P. vivax parasites resistant to chloroquine and the effectiveness of antifolates in vivax malaria treatment together with a correlation between mutations in P. vivax dhfr and dhps genes and SP treatment failure, the point mutations in these genes were investigated., Methods: Blood samples from 54 patients experiencing vivax malaria symptomatic episodes in the Amazonian Region were investigated. Genomic DNA was extracted using a DNA extraction kit (QIAGENTM). Nested polymerase chain reaction (PCR) amplification was carried out followed by Sanger sequencing to detect single nucleotide polymorphisms (SNPs)., Findings: All tested isolates showed non-synonymous mutations in pvdhfr gene: 117N (54/54, 100%) and 58R (25/54, 46%). Double mutant allele 58R/117N (FRTNI, 28%) was the most frequent followed by triple mutant alleles (58R/117N/173L, FRTNL, 11%; 58R/61M/117N, FRMNI, 5% 117N/173L, FSTNL, 4%) and quadruple mutant allele (58R/61M/117N/173L, FRMNL, 2%). A single mutation was observed at codon C383G in pvdhps gene (SGKAV, 48%)., Conclusion: No evidence of molecular signatures associated with P. vivax resistance to SP was observed in the Brazilian samples.

Background: The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination., Objective: Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy., Methods: The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision., Findings: The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR., Conclusion: Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.

It is known that premature elimination of non-parasitized RBCs (nRBCs) plays an important role in the pathogenesis of malarial anemia, in which suicidal death process (eryptosis) of nRBCs has been suggested to be involved. To check this possibility, we investigate eryptosis during infection of P. berghei ANKA in Wistar rats, a malaria experimental model that, similar to human malaria, the infection courses with low parasitemia and acute anemia. As expected, P. berghei ANKA infection was marked by low parasite burdens that reached a mean peak of 3% between days six and nine post-infection and solved spontaneously. A significant reduction of the hemoglobin levels (~ 30%) was also observed on days subsequent to the peak of parasitemia, persisting until day 16 post-infection. In eryptosis assays, it was observed a significant increase in the levels of PS-exposing nRBC, which coincided with the reduction of hemoglobin levels and was positively related to anemia. In addition to PS externalization, eryptosis of nRBC induced by P. berghei infection was characterized by cytoplasm calcium influx, but not caspases activity. These results confirm our previous studies evidencing a pro-eryptotic effect of malaria infection on nRBCs and show that a caspase-independent eryptotic process is implicated in anemia induced by P. berghei ANKA infection in Wistar rats.

Plasmodium falciparum artemisinin-resistant parasites can be evaluated by examining polymorphisms in the kelch ( PfK13 ) domain. A total of 69 samples from patients with falciparum malaria were analyzed. All samples were from areas in states in Brazil where the parasite was endemic: Acre ( n = 14), Amapá ( n = 15), Amazonas ( n = 30), and Pará ( n = 10). After DNA alignment with the 3D7 reference sequence, all samples were found to be wild type. These data provide a baseline for PfK13 and reinforce the pertinence of artemisinin combination therapy in Brazilian areas., (Copyright © 2018 Gomes et al.)

Zoonotic malaria poses a unique problem for malaria control. Autochthonous cases of human malaria in the Atlantic Forest have recently been attributed to Plasmodium simium, a parasite that commonly infects non-human primates in this Brazilian biome. However, due to its close similarity at both the morphological and molecular level to Plasmodium vivax, the diagnosis of P. simium in this region remains problematic. Therefore, a diagnostic assay able to accurately identify P. simium is important for malaria surveillance. Based on mitochondrial genome sequences, primers were designed to amplify a region containing a SNP specific to P. simium. This region can then be digested with the restriction enzyme HpyCH4III, which results in digestion of P. simium sequences, but not of any other malaria parasite. Fifty-two human and monkey blood samples from different regions and infected with different Plasmodium species were used to validate this protocol. This easy and inexpensive tool can be used for the diagnosis of P. simium in non-human primates and human infections from the Atlantic Forest region to monitor zoonotic malaria transmission in Brazil.

In the last decade it has become clear that, similarly to nucleated cells, enucleated red blood cells (RBCs) are susceptible to programmed apoptotic cell death. Erythrocytic apoptosis seems to play a role in physiological clearance of aged RBCs, but it may also be implicated in anemia of different etiological sources including drug therapy and infectious diseases. In malaria, severe anemia is a common complication leading to death of children and pregnant women living in malaria-endemic regions of Africa. The pathogenesis of malarial anemia is multifactorial and involves both ineffective production of RBCs by the bone marrow and premature elimination of non-parasitized RBCs, phenomena potentially associated with apoptosis. In the present overview, we discuss evidences associating erythrocytic apoptosis with the pathogenesis of severe malarial anemia, as well as with regulation of parasite clearance in malaria. Efforts to understand the role of erythrocytic apoptosis in malarial anemia can help to identify potential targets for therapeutic intervention based on apoptotic pathways and consequently, mitigate the harmful impact of malaria in global public health.

Background: Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale., Methods: We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci., Results: We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay., Conclusions: No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).

Background: In Brazil, malaria is endemic in the Amazon River basin and non-endemic in the extra-Amazon region, which includes areas of São Paulo state. In this state, a number of autochthonous cases of malaria occur annually, and the prevalence of subclinical infection is unknown. Asymptomatic infections may remain undetected, maintaining transmission of the pathogen, including by blood transfusion. In these report it has been described subclinical Plasmodium infection in blood donors from a blood transfusion centre in São Paulo, Brazil., Methods: In this cross-sectional study, representative samples of blood were obtained from 1,108 healthy blood donors at the Fundação Pró-Sangue Hemocentro de São Paulo, the main blood transfusion centre in São Paulo. Malaria exposure was defined by the home region (exposed: forest region; non-exposed: non-forest region). Real-time PCR was used to detect Plasmodium falciparum and Plasmodium vivax. Subclinical malaria cases were geo-referenced., Results: Eighty-four (7.41%) blood donors tested positive for Plasmodium; 57 of these were infected by P. falciparum, 25 by P. vivax, and 2 by both. The prevalence of P. falciparum and P. vivax was 5.14 and 2.26, respectively. The overall prevalence ratio (PR) was 3.23 (95% confidence interval (CI) 2.03, 5.13); P. falciparum PR was 16.11 (95% CI 5.87, 44.21) and P. vivax PR was 0.47 (95% CI 0.2, 1.12). Plasmodium falciparum subclinical malaria infection in the Atlantic Forest domain was present in the mountain regions while P. vivax infection was observed in cities from forest-surrounded areas., Conclusions: The presence of Plasmodium in healthy blood donors from a region known as non-endemic, which is important in the context of transfusion biosafety, was described. Infected recipients may become asymptomatic carriers and a reservoir for parasites, maintaining their transmission. Furthermore, P. falciparum PR was positively associated with the forest environment, and P. vivax was associated with forest fragmentation.

A case of autochthonous Plasmodium vivax malaria with sub-microscopic parasitaemia and polyclonal B-cell activation (PBA) (as reflected by positive IgM and IgG serology for toxoplasmosis, cytomegalovirus, and antinuclear and rheumatoid factors) was diagnosed by polymerase chain reaction (PCR) after consecutive negative rapid diagnostic test results and blood films. The patient, a 44-year-old man from Rio de Janeiro state, Brazil, had visited the Atlantic Forest, a tourist, non-malaria-endemic area where no autochthonous cases of 'bromeliad malaria' has ever been described. The characteristic pattern of fever, associated with PBA, was the clue to malaria diagnosis, despite consecutive negative thick blood smears. The study highlights a need for changes in clinical and laboratory diagnostic approaches, namely the incorporation of PCR as part of the current routine malaria diagnostic methods in non-endemic areas.

Background: Immune responses to malaria blood stage infection are in general defective, with the need for long-term exposure to the parasite to achieve immunity, and with the development of immunopathology states such as cerebral malaria in many cases. One of the potential reasons for the difficulty in developing protective immunity is the poor development of memory responses. In this paper, the potential association of cellular reactivity in lymphoid organs (spleen, lymph nodes and Peyer's patches) with immunity and pathology was evaluated during Plasmodium berghei ANKA infection in CBA mice., Methods: CBA mice were infected with 1 x 10(6) P. berghei ANKA-parasitized erythrocytes and killed on days 3, 6-8 and 10 of infection. The spleen, lymph nodes and Peyer's patches were collected, fixed in Carson's formalin, cut in 5 mum sections, mounted in glass slides, stained with Lennert's Giemsa and haematoxylin-eosin and analysed with bright-field microscopy., Results: Early (day 3) strong activation of T cells in secondary lymphoid organs was observed and, on days 6-8 of infection, there was overwhelming activation of B cells, with loss of conventional germinal center architecture, intense centroblast activation, proliferation and apoptosis but little differentiation to centrocytes. In the spleen, the marginal zone disappeared and the limits between the disorganized germinal center and the red pulp were blurred. Intense plasmacytogenesis was observed in the T cell zone., Conclusion: The observed alterations, especially the germinal center architecture disturbance (GCAD) with poor centrocyte differentiation, suggest that B cell responses during P. berghei ANKA infection in mice are defective, with potential impact on B cell memory responses.

Immune responses to malaria infections are characterized by strong T and B cell activation, which, in addition of potentially causing immunopathology, are of poor efficacy against the infection. It is possible that the thymus is involved in the origin of immunopathological reactions and a target during malaria infections. This work was developed in an attempt to further clarify these points. We studied the sequential changes in the thymus of CBA mice infected with Plasmodium berghei ANKA, a model in which 60-90% of the infected animals develop cerebral malaria. During the acute phase of infection, different degrees of thymocyte apoptosis were recorded. (1) starry-sky pattern of diffuse apoptosis with maintenance of cortical-medullary structure; (2) intense apoptosis with cortical atrophy, with absence of large cells; (3) severe cortical thymocyte depletion, resulting in cortical-medullary inversion. In the latter, only residual clusters of small thymocytes were observed within the framework of epithelial cells. The intensity of thymus alterations could not be associated with the degree of parasitemia, the expression of clinical signs of cerebral malaria or intensity of brain lesions. The implications of these events for malaria immunity and pathology are discussed.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Seven of 73 mongrel dogs in Rio de Janeiro gave positive results in skin tests with a polysaccharide mycelial antigen from Histoplasma capsulatum. Five of the positive reactors were necropsied and four of them had disseminated histoplasmosis proved by histopathology and culture. Four healthy puppies exposed for 10 min to soil at the site of a known outbreak of histoplasmosis developed symptoms and died 7-14 days after exposure with fulminant histoplasmosis. These experiments show the value of dogs as epidemiological indicator of histoplasmosis and as experimental models for the disease.

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.

Soluble antigens (Ag) from Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus were prepared and standardized by double immunodiffusion (DID) and immunoelectroosmophoresis (IEOP). No difference in sensitivity was observed between the two techniques; 100% of standard patient sera were positive with P. brasiliensis and A. fumigatus Ag and 83.3% were positive with H. capsulatum Ag. The specificity of the tests was verified testing 96 sera from patients with paracoccidioidomycosis, histoplasmosis, systemic candidiasis, sporotrichosis, tuberculosis, lung cancer, visceral or cutaneous leishmaniasis and 18 sera from healthy individuals. All the three antigens were 100% specific with the DID (using the identification pattern indicated by the confluence of test serum with standard serum precipitin lines as a positive criterium). However in the IEOP, the specificity varied with each Ag. Positive reactions with P. brasiliensis Ag were observed in 16.7% of histoplasmosis sera and in 10% of cutaneous leishmaniasis sera. On the other hand 31.8% of paracoccidioidomycosis and 10% of cutaneous leishmaniasis sera reacted with H. capsulatum Ag. The high sensitivity and specificity of the DID test, its easy reproducibility and low cost, led us to consider it highly appropriate as a routine procedure for the screening of patients with respiratory infections.

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.