Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP1(19), PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP1(19). Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.

Abnormal humoral immune response to influenza vaccination in pediatric type-1 human immunodeficiency virus infected patients receiving highly active antiretroviral therapy

Given that highly active antiretroviral therapy (HAART) has been demonstrated useful to restore immune competence in type-1 human immunodeficiency virus (HIV-1)-infected subjects, we evaluated the specific antibody response to influenza vaccine in a cohort of HIV-1-infected children on HAART so as to analyze the quality of this immune response in patients under antiretroviral therapy. Sixteen HIV-1-infected children and 10 HIV-1 seronegative controls were immunized with a commercially available trivalent inactivated influenza vaccine containing the strains A/H1N1, A/H3N2, and B. Serum hemagglutinin inhibition (HI) antibody titers were determined for the three viral strains at the time of vaccination and 1 month later. Immunization induced a significantly increased humoral response against the three influenza virus strains in controls, and only against A/H3N2 in HIV-1-infected children. The comparison of post-vaccination HI titers between HIV-1+ patients and HIV-1 negative controls showed significantly higher HI titers against the three strains in controls. In addition, post vaccination protective HI titers (defined as equal to or higher than 1:40) against the strains A/H3N2 and B were observed in a lower proportion of HIV-1+ children than in controls, while a similar proportion of individuals from each group achieved protective HI titers against the A/H1N1 strain. The CD4+ T cell count, CD4/CD8 T cells ratio, and serum viral load were not affected by influenza virus vaccination when pre- vs post-vaccination values were compared. These findings suggest that despite the fact that HAART is efficient in controlling HIV-1 replication and in increasing CD4+ T cell count in HIV-1-infected children, restoration of immune competence and response to cognate antigens remain incomplete, indicating that additional therapeutic strategies are required to achieve a full reconstitution of immune functions.

Polymorphism in the promoter region of the mannose-binding lectin gene among human T-cell lymphotropic virus infected subjects

The present study investigated the frequency of the mutations at positions -550 and -221 of the mannose-binding lectin (MBL) gene in a sample of 75 human T-cell lymphotropic virus (HTLV) infected patients and 96 HTLV seronegative controls, in order to evaluate the occurrence of a possible association between the polymorphism and HTLV infection. A sequence specific primer-polymerase chain reaction was used for discrimination of the polymorphism. The analysis of allele frequencies at position -550 did not show any significant differences between HTLV infected group and controls, but there was a significant difference at position -221. The comparative analysis of haplotypes frequencies were not significant, but the genotype frequencies between the two groups, revealed a higher prevalence of genotype LYLX (25.3%), associated with medium and low MBL serum levels among HTLV infected subjects. The odds ratio estimation demonstrated that the presence of genotype LYLX was associated with an increased risk of HTLV infection (p = 0.0096; 1.38 < IC95% < 7.7605). There was no association between proviral load and the promoter polymorphism, but when promoter and exon 1 mutations were matched, it was possible to identify a significant higher proviral load among HTLV infected individuals carrying haplotypes correlated to low serum levels of MBL. The present study shows that the polymorphism in the promoter region of the MBL gene may be a genetic marker associated with HTLV infection, and emphasizes the need for further studies to determinate if the present polymorphism have any impact on diseases linked to HTLV infection.

Characterization of polymorphisms in the mannose-binding lectin gene promoter among human immunodeficiency virus 1 infected subjects

The present study investigated the prevalence of mutations in the -550 (H/L) and -221 (X/Y) mannose-binding lectin (MBL) gene promoter regions and their impact on infection by human immunodeficiency virus 1 (HIV-1) in a population of 128 HIV-1 seropositive and 97 seronegative patients. The allele identification was performed through the sequence-specific primer polymerase chain reaction method, using primer sequences specific to each polymorphism. The evolution of the infection was evaluated through CD4+ T-lymphocyte counts and plasma viral load. The allele and haplotype frequencies among HIV-1-infected patients and seronegative healthy control patients did not show significant differences. CD4+ T-lymphocyte counts showed lower levels among seropositive patients carrying haplotypes LY, LX and HX, as compared to those carrying the HY haplotype. Mean plasma viral load was higher among seropositive patients with haplotypes LY, LX and HX than among those carrying the HY haplotype. When promoter and exon 1 mutations were matched, it was possible to identify a significantly higher viral load among HIV-1 infected individuals carrying haplotypes correlated to low serum levels of MBL. The current study shows that haplotypes related to medium and low MBL serum levels might directly influence the evolution of viral progression in patients. Therefore, it is suggested that the identification of haplotypes within the promoter region of the MBL gene among HIV-1 infected persons should be further evaluated as a prognostic tool for AIDS progression.

Seroepidemiological study of human parvovirus B19 among human immunodeficiency virus-infected patients in a medium-sized city in Rio de Janeiro, Brazil

Parvovirus B19 (B-19) may cause chronic anaemia in immunosuppressed patients, including those infected with human immunodeficiency virus (HIV). We studied single serum samples from 261 consecutive HIV-infected patients using an enzyme immunoassay to detect IgG antibodies to B-19. The seroprevalence of B-19-IgG was 62.8%. The differences in seroprevalence across gender, age, educational categories, year of collection of the serum samples, clinical and antiretroviral therapy characteristics, CD4+ count, CD4+ and CD8+ percentage and CD4+/CD8+ ratios were neither substantial nor statistically significant. There was a non-significant, inverse association between B-19 seropositivity and plasma HIV load and haemoglobin level. Our results indicated that 37.1% of patients might be susceptible to B-19 infection and remained at risk for being infected, mainly during epidemic periods. As B-19 infection can be treated with immune globulin preparations, it may be included in the diagnostic approach toward chronic anaemia in HIV-infected patients.

Laboratorial atopy markers in children with human immunodeficiency virus

Changes in immune system functions are one of the most important consequences of human immunodeficiency virus (HIV) infection. Studies have reported a higher prevalence of disease mediated by immunological hypersensitivity mechanisms in HIV-positive patients. This study aims to observe how immunological changes in HIV-infected children interfere in atopy determinants. Fifty-seven HIV-positive children were studied between June 2004-August 2005 to evaluate the possible modifications in atopy diagnosis from prick test environmental allergen reactivity. Patients were subjected to two evaluations: on both occasions, atopic and non-atopic groups were correlated with immunological (CD4+ and CD8+ lymphocyte concentrations and serum levels of IgA, IgM, IgG and IgE) and viral parameters (HIV viral load). The percent atopy was 20.05 in the first and 29.82 in the second evaluation and atopy was diagnosed in patients without immunosuppression or with moderate immunosuppression. Six patients changed from a negative to a positive atopy profile. One patient with a decreased CD4+ T lymphocyte concentration failed to demonstrate prick test positivity between evaluations. Multivariate analysis showed that the variables associated with atopy diagnosis included a personal history of allergic diseases as well as elevated IgE for age and elevated IgE levels. Atopy development in HIV-infected children seems to be modulated by genetic and environmental factors as well as immunological condition.

Detection of human herpesvirus 7 infection in young children presenting with exanthema subitum

In this study, we assessed the prevalence of human herpesvirus-7 (HHV-7) in 141 serum samples from children less than four years of age with exanthematic disease. All samples were negative for measles, rubella, dengue fever and parvovirus B19 infection. Testing for the presence of human herpesvirus-6 (HHV-6)-specific high avidity IgG antibodies by indirect immunofluorescence assay (IFA) revealed two main groups: one composed of 57 patients with recent primary HHV-6 infection and another group of 68 patients showing signs of past HHV-6 infection. Another 16 samples had indeterminate primary HHV-6 infection, by both IgG IFA and IgM IFA. Serum samples were subjected to a nested polymerase chain reaction to detect the presence of HHV-7 DNA. Among patients with a recent primary HHV-6 infection, HHV-7 DNA was present in 1.7% of individuals; however, 5.8% of individuals tested positive for HHV-7 DNA in the group with past primary HHV-6 infection. Among the 16 samples with indeterminate diagnosis, 25% (4/16) had HHV-7 DNA (p < 0.002). We hypothesise that HHV-7 might be the agent that causes exanthema. However, a relationship between clinical manifestations and the detection of virus DNA does not always exist. Therefore, a careful interpretation is necessary to diagnose a primary infection or a virus-associated disease. In conclusion, we detected HHV-7 DNA in young children from the state of Rio de Janeiro, Brazil.

Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing

Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 µg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85% and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.

Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

A new survey of the serology of human Trypanosoma cruziinfection in the Rio Negro microregion, Brazilian Amazon: a critical analysis

The serology of human Trypanosoma cruziinfection in the Rio Negro microregion is very complex because of the large numbers of false-positive cases that result from low antibody titres and cross-reactions with other infections. In the present study, we collected 4,880 blood samples on filter paper; of these, indirect immunofluorescence (IIF) was strongly reactive in 221 (4.5%), which were considered to be positive (IIF strongly reactive; high intensity of fluorescence) and weakly reactive in 302 (6.2%), which were considered to be doubtful (IIF weakly reactive; low intensity of fluorescence). The confirmatory test on the serum using at least two of three techniques (IIF, conventional ELISA and recombinant ELISA) on 137 samples that were positive in the screening test only confirmed 33 cases (24.1%). Of the 178 samples that were considered doubtful in the screening test, only 10 (5.6%) were considered to be positive in the confirmatory test. Finally, we recommend that the serological diagnosis of T. cruziinfection in the Amazon region be made using at least two different techniques, for example immunofluorescence and ELISA and confirmed by Western blot analysis when possible.

Clinical features and laboratory findings of human parvovirus B19 in human immunodeficiency virus-infected patients

Immunocompromised patients may develop severe chronic anaemia when infected by human parvovirus B19 (B19V). However, this is not the case in human immunodeficiency virus (HIV)-infected patients with good adherence to highly active antiretroviral treatment (HAART). In this study, we investigated the clinical evolution of five HIV-infected patients receiving HAART who had B19V infections confirmed by serum polymerase chain reaction. Four of the patients were infected with genotype 1a strains and the remaining patient was infected with a genotype 3b strain. Anaemia was detected in three of the patients, but all patients recovered without requiring immunoglobulin and/or blood transfusions. In all cases, the attending physicians did not suspect the B19V infections. There was no apparent relationship between the infecting genotype and the clinical course. In the HAART era, B19V infections in HIV-positive patients may be limited, subtle or unapparent.

Experimental infection with the Toxoplasma gondii ME-49 strain in the Brazilian BR-1 mini pig is a suitable animal model for human toxoplasmosis

Toxoplasma gondii causes toxoplasmosis, a worldwide disease. Experimentation with pigs is necessary for the development of new therapeutic approaches to human diseases. BR-1 mini pigs were intramuscularly infected with T. gondii with tachyzoites (RH strain) or orally infected with cysts (ME-49 strain). Haematology and serum biochemistry were analysed and buffy coat cells were inoculated in mice to determine tachyzoite circulation. No alterations were observed in erythrocyte and platelet values; however, band neutrophils increased seven days after infection with ME-49. Serology of the mice inoculated with pig blood leucocytes revealed circulating ME-49 or RH strain tachyzoites in the pigs' peripheral blood at two and seven or nine days post-infection. The tachyzoites were also directly observed in blood smears from the infected pigs outside and inside leucocytes for longer periods. Alanine-aminotransferase was high at days 21 and 32 in the RH infected pigs. After 90 days, the pigs were euthanised and their tissue samples were processed and inoculated into mice. The mice serology revealed the presence of parasites in the hearts, ileums and mesenteric lymph nodes of the pigs. Additionally, cysts in the mice were only observed after pig heart tissue inoculation. The infected pigs presented similar human outcomes with relatively low pathogenicity and the BR-1 mini pig model infected with ME-49 is suitable to monitor experimental toxoplasmosis.

Genetic variability in G2 and F2 region between biological clones of human respiratory syncytial virus with or without host immune selection pressure

Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child's serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.