Respiratory viruses in the pediatric intensive care unit: prevalence and clinical aspects

A survey was conducted in two pediatric intensive care units in hospitals in Porto Alegre, Brazil, in order to monitor the main respiratory viruses present in bronchiolitis and/or pneumonia and their involvement in the severity of viral respiratory infections. Viral respiratory infection prevalence was 38.7%. In bronchiolitis, respiratory syncytial virus (RSV) was detected in 36% of the cases. In pneumonia, the prevalence rates were similar for adenovirus (10.3%) and RSV (7.7%). There was a difference among the viruses detected in terms of frequency of clinical findings indicating greater severity. Frequency of crackles in patients with RSV (47.3%) showed a borderline significance (p = 0.055, Fisher's exact test) as compared to those with adenovirus (87.5%). The overall case fatality rate in this study was 2.7%, and adenovirus showed a significantly higher case fatality rate (25%) than RSV (2.8%) (p = 0.005). Injected antibiotics were used in 49% of the children with RSV and 60% of those with adenovirus. Adenovirus was not detected in any of the 33 children submitted to oxygen therapy.

The human immunodeficiency virus preventive vaccine research at the French National Agency for acquired immunodeficiency syndrome research

The human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic is of unprecedented gravity and is spreading rapidly, notably in the most disadvantaged regions of the world. The search for a preventive vaccine is thus an absolute priority. For over 10 years the French National Agency for AIDS research (ANRS) has been committed to an original program combining basic science and clinical research. The HIV preventive vaccine research program run by the ANRS covers upstream research for the definition of immunogens, animal models, and clinical research to evaluate candidate vaccines. Most researchers in 2004 believe that it should be possible to obtain partial vaccine protection through the induction of a strong and multiepitopic cellular response. Since 1992, the ANRS has set up 15 phases I and II clinical trials in order to evaluate the safety and the capacity of the candidate vaccines for inducing cellular immune responses. The tested candidate vaccines were increasingly complex recombinant canarypox viruses (Alvac) containing sequences coding for certain viral proteins, utilized alone or combined with other immunogens (whole or truncated envelope proteins). ANRS has also been developing an original strategy based on the utilization of lipopeptides. These comprise synthetic fragments of viral proteins associated with lipids that facilitate the induction of a cellular immune response. These approaches promptly allowed the assessment of a prime-boost strategy combining a viral vector and lipopeptides.

Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Frequency of viruses associated with acute respiratory infections in children younger than five years of age at a locality of Mexico City

A locality in the district of Tlalpan, Mexico City, was selected in order to identify the viral agents in children younger than 5 years of age with acute respiratory infection (ARI). A total of 300 children were randomly selected and were included in this study for a period of 13 months. During this period nasopharyngeal exudates were collected for the isolation of viral agents. Monoclonal fluorescent antibodies were used for viral identification after cell culture. Viral infection was detected in 65% of the specimens. The respiratory syncytial virus (RSV) was the most common virus agent detected. Children required an average of two consultations during the study period. Two high incidence peaks were observed, one during the summer and the other during winter; the most frequent viruses during these seasons were influenza A and RSV, respectively. The largest number of viruses was isolated in the group of children between 1 and 2 years of age and in the group between 4 and 5 years of age. This study demonstrated the presence of ARI and of different viruses in a period of 13 months, as well as the most frequent viruses in children younger than 5 years of age from a community of Mexico City.

Respiratory viruses in children younger than five years old with acute respiratory disease from 2001 to 2004 in Uberlândia, MG, Brazil

The main viruses involved in acute respiratory diseases among children are: respiratory syncytial virus (RSV), influenzavirus (FLU), parainfluenzavirus (PIV), adenovirus (AdV), human rhinovirus (HRV), and the human metapneumovirus (hMPV). The purpose of the present study was to identify respiratory viruses that affected children younger than five years old in Uberlândia, Midwestern Brazil. Nasopharyngeal aspirates from 379 children attended at Hospital de Clínicas (HC/UFU), from 2001 to 2004, with acute respiratory disease, were collected and tested by immunofluorescence assay (IFA) to detect RSV, FLU A and B, PIV 1, 2, and 3 and AdV, and RT-PCR to detect HRV. RSV was detected in 26.4% (100/379) of samples, FLU A and B in 9.5% (36/379), PIV 1, 2 and 3 in 6.3% (24/379) and AdV in 3.7% (14/379). HRV were detected in 29.6% (112/379) of the negative and indeterminate samples tested by IFI. RSV, particularly among children less than six months of life, and HRV cases showed highest incidence. Negative samples by both IFA and RT-PCR might reflect the presence of other pathogens, such as hMPV, coronavirus, and bacteria. Laboratorial diagnosis constituted an essential instrument to determine the incidence of the most common viruses in respiratory infections among children in this region.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Protection against hepatitis B by the Butang® recombinant vaccine in newborn children in South Brazil

The prevention of hepatitis B by vaccination is one of the most efficient tools to avoid the transmission of the virus. This study evaluated the immunogenicity of the national vaccine Butang® in children born in Campo Mourão City, state of Paraná, Brazil, aged 7 to 12 months, by determining the anti-HBsAg antibodies levels after completion of the National Immunization Program Protocol for hepatitis B. All 70 children evaluated by the MEIA method (immune-enzymatic micro particles) showed seroconversion to the Butang® vaccine. Nine children (12.9%) presented a low response, with anti-HBs titers between 11 and 100 mUI/ml; 39 children (55.7%) showed a good response to the vaccine, with titers between 101 and 1000 mUI/ml; and 22 children (31.4%) showed antibodies titers higher than 1000 mUI/ml. The mean titer of the anti-HBs antibody titers was 1408.1 ± 2870.26 mUI/ml (15.7 to 19560.0 mUI/ml). The levels of antibodies produced by the prematurely-born children were not statistically different from those found in the newborns. Fifty-five children were also evaluated through the ELFA method (ELISA with a final detection in fluorescence), which presented similar results. The results obtained in our study corroborated the effectiveness of the national vaccine Butang® in newborn children of Campo Mourão City, Paraná, even if they were premature.

Cell proliferation and interferon-gamma response to recombinant MBP-3, NarL, MT-10.3, and 16kDa Mycobacterium tuberculosis antigens in Brazilian tuberculosis patients

Human pulmonary tuberculosis (TB) is a worldwide public health problem. In resistant individuals, control of the infection mainly requires development of a Th1 cell immune response with production of cytokines, of which interferon-gamma (IFN-gamma)plays an important role. Several antigens from Mycobacterium tuberculosis complex has been described for use in vaccine development or for diagnostic purposes, however little evaluation has been done in endemic area for TB. The proliferative and IFN-gamma human T cell immune responses, to four recombinant proteins (MBP-3, NarL, MT-10.3, 16 kDa) and PPD, of 38 Brazilian TB patients (6 untreated and 32 treated) and 67 controls (38 positive and 29 negative tuberculin skin test - TST) were compared. The highest reactivity mean rate was obtained with PPD followed by 16 kDa in TB patients. While most of the patients (87%) and controls (> 64%) respond to the PPD, 16kDa was more specifically recognized (> 21%) although less sensitive (54%). When TB patients were divided according to treatment status, opposite to PPD, higher average level of IFN-gamma was induced by 16kDa in untreated (505 pg/ml) compared to treated TB patients and TST+ (269.8 pg/ml x 221.6pg/ml, respectively), although the difference was not significant. These data show that in contrast with the other recombinant proteins, the stimulatory potency of 16kDa to induce proliferative and INF-gamma response was more effective and is more recognized by active TB untreated patients, eliciting in control individuals a more selective immune response than PPD.

Re-mapping the molecular features of the human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1 Brazilian sequences using a bioinformatics unit established in Salvador, Bahia, Brazil, to give support to the viral epidemiology studies

The analysis of genetic data for human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type 1 (HTLV-1) is essential to improve treatment and public health strategies as well as to select strains for vaccine programs. However, the analysis of large quantities of genetic data requires collaborative efforts in bioinformatics, computer biology, molecular biology, evolution, and medical science. The objective of this study was to review and improve the molecular epidemiology of HIV-1 and HTLV-1 viruses isolated in Brazil using bioinformatic tools available in the Laboratório Avançado de Sáude Pública (Lasp) bioinformatics unit. The analysis of HIV-1 isolates confirmed a heterogeneous distribution of the viral genotypes circulating in the country. The Brazilian HIV-1 epidemic is characterized by the presence of multiple subtypes (B, F1, C) and B/F1 recombinant virus while, on the other hand, most of the HTLV-1 sequences were classified as Transcontinental subgroup of the Cosmopolitan subtype. Despite the high variation among HIV-1 subtypes, protein glycosylation and phosphorylation domains were conserved in the pol, gag, and env genes of the Brazilian HIV-1 strains suggesting constraints in the HIV-1 evolution process. As expected, the functional protein sites were highly conservative in the HTLV-1 env gene sequences. Furthermore, the presence of these functional sites in HIV-1 and HTLV-1 strains could help in the development of vaccines that pre-empt the viral escape process.

Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19)-PADRE). In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin (LT) developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.

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.

The epidemiology and antigenic characterization of influenza viruses isolated in Curitiba, South Brazil

Several studies conducted all over the world have reported that the influenza virus is associated with great morbidity and mortality rates. In this study, we analyzed the incidence of the influenza virus between 2000 and 2003 in Curitiba. We studied 1621 samples obtained from outpatients and hospitalized patients of both sexes and all ages. The study was conducted at the local primary care health units (outpatients) and at the tertiary care unit (hospitalized) of the General Hospital of the Federal University in the state of Paraná, Brazil. Nasopharyngeal aspirates and, eventually, bronchoalveolar lavage were assayed for the presence of viral antigens, either by indirect immunofluorescence or cell culture. Of the samples studied, 135 (8.3%) were positive for influenza virus, and of those, 103 (76.3%) were positive for type A and 32 (23.7%) for type B. Additionally, positive samples were analyzed by reverse transcription followed by polymerase chain reaction and subtypes H1 and H3 were identified from this group. A high incidence of positive samples was observed mainly in the months with lower temperatures. Furthermore, outpatients showed a higher incidence of influenza viruses than hospitalized patients.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.