Identification of Human T-lymphotropic Virus Type I (HTLV-I) Subtypes Using Restrited Fragment Length Polymorphism in a Cohort of Asymptomatic Carriers and Patients with HTLV-I-associated Myelopathy/tropical Spastic Paraparesis from São Paulo, Brazil

Although human T-lymphotropic virus type I (HTLV-I) exhibits high genetic stability, as compared to other RNA viruses and particularly to human immunodeficiency virus (HIV), genotypic subtypes of this human retrovirus have been characterized in isolates from diverse geographical areas. These are currently believed not to be associated with different pathogenetic outcomes of infection. The present study aimed at characterizing genotypic subtypes of viral isolates from 70 HTLV-I-infected individuals from São Paulo, Brazil, including 42 asymptomatic carriers and 28 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), using restricted fragment length polymorphism (RFLP) analysis of long terminal repeat (LTR) HTLV-I proviral DNA sequences. Peripheral blood mononuclear cell lysates were amplified by nested polymerase chain reaction (PCR) and amplicons submitted to enzymatic digestion using a panel of endonucleases. Among HTLV-I asymptomatic carriers, viral cosmopolitan subtypes A, B, C and E were identified in 73.8%, 7.1%, 7.1% and 12% of tested samples, respectively, whereas among HAM/TSP patients, cosmopolitan A (89.3%), cosmopolitan C (7.1%) and cosmopolitan E (3.6%) subtypes were detected. HTLV-I subtypes were not statistically significant associated with patients' clinical status. We also conclude that RFLP analysis is a suitable tool for descriptive studies on the molecular epidemiology of HTLV-I infections in our environment.

Protease activity in Giardia duodenalis trophozoites of axenic strains isolated from symptomatic and asymptomatic patients

We have examined by gelatin-SDS-PAGE the protease activity in cell lysates of Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic patient (BTU-11) and an asymptomatic carrier (BTU-10), and the reference strain Portland 1 (P1). The proteolysis band patterns showed differences among strains isolated from asymptomatic and symptomatic individuals. The lysate of the strain BTU-10, showed only five hydrolysis bands, while a greater number of bands (10-11 bands) was seen in strains BTU-11 and P1. The protease activity in all lysates was inhibited by cysteine (E-64 and iodoacetamide) and serine proteases (TPCK and TLCK) inhibitors, but not by PMSF and EDTA. In general, the results revealed protease activities in G. duodenalis trophozoites of Brazilian axenic strains and the predominance of cysteine proteinases. It should be stressed the inter-strain difference in hydrolysis band patterns observed between strains isolated from symptomatic patients and the strain obtained from an asymptomatic carrier.

Increased plasma levels of tumor necrosis factor-alpha in asymptomatic/"indeterminate" and Chagas disease cardiomyopathy patients

We compared plasma tumor necrosis factor-alpha (TNF-alpha) levels among asymptomatic/"indeterminate" Chagas disease patients (ASY) and patients across the clinical spectrum of chronic Chagas disease cardiomyopathy (CCC). Idiopathic dilated cardiomyopathy (DCM) patients and normal controls (NC) were included as controls. ASY Chagas disease patients had significantly higher plasma TNF-alpha levels than NC. TNF-alpha levels among severe CCC patients with significant left ventricular (LV) dysfunction were similar to those of DCM patients, showing average 2-fold higher levels than CCC patients without LV dysfunction and ASY patients, and 8-fold higher levels than NC. In Chagas disease, chronic TNF-a production prior to heart failure may play a role in CCC progression.

A new challenge for malaria control in Brazil: asymptomatic Plasmodium infection - a review

The evolution of malaria in Brazil, its morbidity, the malaria control programs, and the new challenges for these programs in the light of the emergence of asymptomatic infection in the Amazon region of Brazil were reviewed. At least six Brazilian research groups have demonstrated that asymptomatic infection by Plasmodium is an important impediment to malaria control, among mineral prospectors in Mato Grosso and riverside communities in Rondônia and, in our group, in the middle and upper reaches of the Negro river, in the state of Amazonas. Likewise, other researchers have studied the problem among indigenous communities in the Colombian, Peruvian, and Venezuelan parts of the Amazon basin, adjacent to Brazil. The frequency of positive results from the polymerase chain reaction (PCR) among asymptomatic individuals has ranged from 20.4 to 49.5%, and the presence of Plasmodium in the thick blood smears, from 4.2 to 38.5%. Infection with Anopheles darlingi has also been demonstrated by xenodiagnosis among asymptomatic patients with positive PCR results. If a mean of 25% is taken for the asymptomatic infection caused by Plasmodium sp. in the Amazon region of Brazil, malaria control will be difficult to achieve in that region with the measures currently utilized for such control.

Early infection and asymptomatic spread of hepatitis A virus in a public child care center in Rio de Janeiro, Brazil: should attending children under two years of age be vaccinated?

A cross-sectional study was conducted in order to identify hepatitis A virus (HAV) serological markers in 418 individuals (mean age, 16.4 years; range, 1 month-80 years) at a public child care center in Rio de Janeiro, Brazil, as well as to analyze risk factors and determine circulating genotypes. Serum samples were tested using an enzyme immunoassay. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect and characterize HAV RNA, and sequencing was performed. Anti-HAV antibodies and IgM anti-HAV antibodies were detected, respectively, in 89.5% (374/418) and 10.5% (44/418) of the individuals tested. Acute HAV infection in children was independently correlated with crawling (p < 0.05). In 56.8% (25/44) of the IgM anti-HAV-positive individuals and in 33.3% (5/15) of the IgM anti-HAV-negative individuals presenting clinical symptoms, HAV RNA was detected. Phylogenetic analysis revealed co-circulation of subgenotypes IA and IB in 93.3% (28/30) of the amplified samples. In present study, we verify that 79% (30/38) of children IgM anti-HAV-positive were asymptomatic. In child care centers, this asymptomatic spread is a more serious problem, promoting the infection of young children, who rarely show signs of infection. Therefore, vaccinating children below the age of two might prevent the asymptomatic spread of hepatitis A.

Urban malaria in the Brazilian Western Amazon Region I: high prevalence of asymptomatic carriers in an urban riverside district is associated with a high level of clinical malaria

Cross sectional studies on malaria prevalence was performed in 2001, 2002, and 2004 in Vila Candelária, an urban riverside area of Porto Velho, Rondônia, in the Brazilian Western Amazon, followed by longitudinal surveys on malaria incidence. Vila Candelária is a working class district, provided with electricity, water supply, and basic sanitation. Previous preliminary surveys indicated high malaria incidence in this community. At the end of year 2000 regular diagnostic and treatment measures for malaria were introduced, with active search of febrile cases among residents. Despite of both rapid treatment of cases and relative good sanitary and housing conditions, the malaria incidence persisted at high levels during the following years with an annual parasite index of 150 to 300/1000 inhabitants. Parasite surveys in 2001, 2002, and 2004 achieved through microscopy and polymerase chain reaction to diagnose malaria showed a constant high prevalence of asymptomatic carriers for both Plasmodium falciparum and P. vivax parasites. It was concluded that asymptomatic carriers represent an important reservoirs of parasites and that the carriers might contribute to maintaining the high level of transmission. Comparing our findings to similar geo-demographic situations found in other important urban communities of the Brazilian Amazon, we propose that asymptomatic carriers could explain malaria's outbreaks like the one recently observed in Manaus.

Clinical spectrum of uncomplicated malaria in semi-immune Amazonians: beyond the " symptomatic " vs " asymptomatic " dichotomy

We analyzed prospectively 326 laboratory-confirmed, uncomplicated malarial infections (46.3% due to Plasmodium vivax, 35.3% due to P. falciparum, and 18.4% mixed-species infections) diagnosed in 162 rural Amazonians aged 5-73 years. Thirteen symptoms (fever, chills, sweating, headache, myalgia, arthralgia, abdominal pain, nausea, vomiting, dizziness, cough, dyspnea, and diarrhea) were scored using a structured questionnaire. Headache (59.8%), fever (57.1%), and myalgia (48.4%) were the most frequent symptoms. Ninety-six (29.4%) episodes, all of them diagnosed during cross-sectional surveys of the whole study population (96.9% by molecular technique only), were asymptomatic. Of 93 symptom-less infections left untreated, only 10 became symptomatic over the next two months following diagnosis. Fever was perceived as " intense " in 52.6% of 230 symptomatic malaria episodes, with no fever reported in 19.1% episodes although other symptoms were present. We found significant differences in the prevalence and perceived intensity of fever and other clinical symptoms in relation to parasite load at the time of diagnosis and patient's age, cumulative exposure to malaria, recent malaria morbidity, and species of malaria parasite. These factors are all likely to affect the effectiveness of malaria control strategies based on active or passive detection of febrile subjects in semi-immune populations.

Asymptomatic Plasmodium spp. infection in Tierralta, Colombia

With the aim of determining the prevalence of asymptomatic Plasmodium spp. infection by thick smear and PCR and its association with demographic and epidemiological characteristics in the village of Nuevo Tay, Tierralta, Córdoba, Colombia, a cross-sectional population study was carried out, using random probabilistic sampling. Venous blood samples were taken from 212 people on day 0 for thick smear and PCR. Clinical follow-up and thick smears were carried out on days 14 and 28. The prevalence of Plasmodium spp. infection was 17.9% (38/212; 95% CI: 12.5-23.3%) and the prevalence of asymptomatic Plasmodiumspp. infection was 14.6% (31/212; 95% CI: 9.6-19.6%). Plasmodium vivax was found more frequently (20/31; 64.5%) than Plasmodium falciparum (9/31; 29%) and mixed infections (2/31; 6.5%). A significantly higher prevalence of asymptomatic infection was found in men (19.30%) than in women (9.18%) (prevalence ratio: 2.10; 95% CI: 1.01-4.34%; p = 0.02). People who developed symptoms had a significantly higher parasitemia on day 0 than those who remained asymptomatic, of 1,881.5 ± 3,759 versus 79 ± 106.9 (p = 0.008). PCR detected 50% more infections than the thick smears. The presence of asymptomatic Plasmodium spp. infection highlights the importance of carrying out active searches amongst asymptomatic populations residing in endemic areas.

Efficacy of benznidazol treatment for asymptomatic chagasic patients from state of Rio Grande do Sul evaluated during a three years follow-up

The efficacy of benznidazol on the treatment of chagasic patients from the state of Rio Grande do Sul was evaluated during a three-year follow-up. A cohort of 80 asymptomatic chronic chagasic patients or blood bank donors (49 male and 31 female) was studied. Their ages varied from 17-42 years, with a mean and a median of 30 and 35 years, respectively. The 80 patients presented positive serology, hemoculture and polymerase chain reaction (PCR). They were treated with 5 mg/Kg benznidazol twice a day for 60 days. Serological, parasitological and PCR methods were used to evaluate response. Serology was performed using commercial ELISA and indirect immunofluorescence (IFI) tests, parasitemia was monitored by hemoculture in LIT medium and PCR with primers S35/S36 was used to amplify a Trypanosoma cruzi 330 bp kDNA repetitive sequence. PCR positivity of 240 seropositive individuals was compared using DNA preparations from whole blood/guanidine EDTA (GE), buffy-coat/GE and frozen buffy-coat. Fifty non-chagasic individuals were used as negative controls. PCR positivity was 86.7% for the frozen buffy-coat, 71.7% for the GE/buffy-coat and 69.2% for the GE/whole blood. The hemocultures became negative just after treatment and remained negative during the three years of follow-up. In the third year after treatment, 9/80 (11.3%) patients presented negative PCR and, from those, four also presented negative serological tests. Furthermore, a reduction in three serological titers was observed in 27/80 (33.8%) of the patients treated. Taken together, the results show that four of the 80 (5.0%) chronic chagasic patients from the state of Rio Grande do Sul were cured after treatment with benznidazol.

Asymptomatic infection with Plasmodium falciparum and Plasmodium vivax in the Brazilian Amazon Basin: to treat or not to treat?

In this study, we determined whether the treatment of asymptomatic parasites carriers (APCs), which are frequently found in the riverside localities of the Brazilian Amazon that are highly endemic for malaria, would decrease the local malaria incidence by decreasing the overall pool of parasites available to infect mosquitoes. In one village, the treatment of the 19 Plasmodium falciparum-infected APCs identified among the 270 residents led to a clear reduction (Z = -2.39, p = 0.017) in the incidence of clinical cases, suggesting that treatment of APCs is useful for controlling falciparum malaria. For vivax malaria, 120 APCs were identified among the 716 residents living in five villages. Comparing the monthly incidence of vivax malaria in two villages where the APCs were treated with the incidence in two villages where APCs were not treated yielded contradictory results and no clear differences in the incidence were observed (Z = -0.09, p = 0.933). Interestingly, a follow-up study showed that the frequency of clinical relapse in both the treated and untreated APCs was similar to the frequency seen in patients treated for primary clinical infections, thus indicating that vivax clinical immunity in the population is not species specific but only strain specific.

Upregulation of hsa-miR-125b in HTLV-1 asymptomatic carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis patients

The retrovirus human T lymphotropic virus type 1 (HTLV-1) promotes spastic paraparesis, adult T cell leukaemia and other diseases. Recently, some human microRNAs (miRNAs) have been described as important factors in host-virus interactions. This study compared miRNA expression in control individuals, asymptomatic HTLV-1 carriers and HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis patients. The proviral load and Tax protein expression were measured in order to characterize the patients. hsa-miR-125b expression was significantly higher in patients than in controls (p = 0.0285) or in the HAM group (p = 0.0312). Therefore, our findings suggest that miR-125b expression can be used to elucidate the mechanisms of viral replication and pathogenic processes.

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.

Variation in haematological parameters in children less than five years of age with asymptomatic Plasmodium infection: implication for malaria field studies

During the season of high malaria transmission, most children are infected by Plasmodium, which targets red blood cells (RBCs), affecting haematological parameters. To describe these variations, we examined the haematological profiles of two groups of children living in a malaria-endemic area. A cross-sectional survey was conducted at the peak of the malaria transmission season in a rural area of Burkina Faso. After informed consent and clinical examination, blood samples were obtained from the participants for malaria diagnosis and a full blood count. Of the 414 children included in the analysis, 192 were not infected with Plasmodium, whereas 222 were asymptomatic carriers of Plasmodium infection. The mean age of the infected children was 41.8 months (range of 26.4-57.2) compared to 38.8 months (range of 22.4-55.2) for the control group (p = 0.06). The asymptomatic infected children tended to have a significantly lower mean haemoglobin level (10.8 g/dL vs. 10.4 g/dL; p < 0.001), mean lymphocyte count (4592/µL vs. 5141/µL; p = 0.004), mean platelet count (266 x 103/µL vs. 385 x 103/µL; p < 0.001) and mean RBC count (4.388 x 106/µL vs. 4.158 x 106/µL; p < 0.001) and a higher mean monocyte count (1403/µL vs. 1192/µL; p < 0.001) compared to the control group. Special attention should be applied when interpreting haematological parameters and evaluating immune responses in asymptomatic infected children living in malaria-endemic areas and enrolled in vaccine trials.

Asymptomatic infection in individuals from the municipality of Barcelos (Brazilian Amazon) is not associated with the anti-Plasmodium falciparum glycosylphosphatidylinositol antibody response

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.

Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.