Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ß-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ß-hematin synthesis (IßHS) and hemoglobin proteolysis (IHbP) in vitro. The ß-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5%, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 µM and 14.4 ± 1.4 µM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0% and 55.3 ± 3.6%, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia (%P) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0% compared to 26.6 ± 3.7%, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.

Pandemic influenza A/H1N1 vaccination coverage, adverse reactions, and reasons for vaccine refusal among medical students in Brazil

The aim of this cross-sectional study was to determine, among medical students at a public university in Rio de Janeiro, Brazil, the acceptance of the pandemic influenza A/H1N1 vaccine during the 2010 mass immunization campaign and the vaccine safety in this group and, among unvaccinated students, the reasons for refusing vaccination. Of a total of 858 students, 678 (79%) participated in the study. Vaccination coverage was 60.4% among students aged 20 to 39 years (an age group targeted for vaccination) and 43.8% among those who did not belong to this age group. The most frequent adverse reactions to the vaccine were pain at the injection site (8.7%) and fever (7.9%). There were no serious adverse reactions. Among students aged 20 to 39 years, the most common reasons for refusing the vaccine were "lack of time" (42.4%), "fear of adverse reactions" (41.9%), and "difficult access to the vaccine" (11.5%). Other reasons for vaccine refusal were "uncertainties about vaccine safety and efficacy" and "vaccination was not needed". To increase the acceptance of the influenza vaccine, a comprehensive immunization program should be offered to these students.

ROTAVIRUS GENOTYPES CIRCULATING IN BRAZIL, 2007-2012: IMPLICATIONS FOR THE VACCINE PROGRAM

SUMMARY Regarding public health in Brazil, a new scenario emerged with the establishment of universal rotavirus (RV) vaccination programs. Herein, the data from the five years of surveillance (2007-2012) of G- and P-type RV strains isolated from individuals with acute gastroenteritis in Brazil are reported. A total of 6,196 fecal specimens were investigated by ELISA and RT-PCR. RVs were detected in 19.1% (1,181/6,196). The peak of RV incidence moved from June-August to September. RV was detected less frequently (19.5%) among children ≤ 5 years than in older children and adolescents (6-18 years) (40.6%). Genotype distribution showed a different profile for each year: G2P[4] strains were most prevalent during 2007-2010, G9P[8] in 2011, and G12P[8] in 2012. Mixed infections (G1+G2P[4], G2+G3P[4]+P[8], G2+G12P[8]), unusual combinations (G1P[4], G2P[6]), and rare strains (G3P[3]) were also identified throughout the study period. Widespread vaccination may alter the RV seasonal pattern. The finding of RV disease affecting older children and adolescents after vaccine implementation has been reported worldwide. G2P[4] emergence most likely follows a global trend seemingly unrelated to vaccination, and G12, apparently, is emerging in the Brazilian population. The rapidly changing RV genotype patterns detected during this study illustrate a dynamic population of co-circulating wildtype RVs in Brazil.

Evaluation of a new vaccine based on pDNA and recombinant protein against Helicobacter pylori

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

PARACOCCIDIOIDOMYCOSIS: CHALLENGES IN THE DEVELOPMENT OF A VACCINE AGAINST AN ENDEMIC MYCOSIS IN THE AMERICAS

SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

The vaccination of h uman beings with live avirulent vaccine of Trypanosoma cruzi: A two year follow-up of the first two cases

A two year follow-up of the first two volunteers vaccinated icith the live PF Trypanosoma cruzi strain demonstrated that the parasitological and clinical test were negative during and after that period. Of the serological tests employed, the CFT presented, in only one case, conflicting results, particulary in one laboratory and among different laboratories. However negative results were greater than all doubtful and positive one combined. The IFT icere negative in both patients. Some comments are made about the sensibility and specificity of these tests. The author concluded that the vaccine, in spite of the very large dose used in these cases, seems to be safe for human beings.

Re-evaluation of the basic procedures involved in the storage of measles vaccine in public health units of the municipality of Niterói, state of Rio de Janeiro, Brazil

Four years after the first visit seventeen public health units were visited again and evaluated as to standards of storage recommended by the Brazilian Immunization Programme. In 100% of the units, refrigerators and proper inside location of vaccines in the refrigerator were adequatety or regularfy maintained and checked, respectively. However, when control of temperature was checked, onfy 64.7% presented adequate storage conditions. In 94.1 % of the units, health workers complained of lack of immediate technical support in emergency situations. In 55.2 % the titers vaccine samples of were under the minimal recommended potency. It is necessary that the factors concerning the cold chain be continualfy evaluated so that the quality of the vaccines that will be used is not affected.

SPf66 vaccine trial in Brazil: conceptual framework study design and analytical approach

This paper describes the study population and the study design of the phase III field trial of the SPf66 vaccine in Brazil. Assessment of validity and precision principles necessary for the appropriate evaluation of the protective effect of the vaccine are discussed, as well as the results of the preliminary analyses of the gathered data. The analytical approach for the estimation of the protective effect of the vaccine is presented. This paper provides the conceptual framework for future publications.

Safety evaluation of SPF66 malaria vaccine in Brazil

The frequency and description of side effects secondaiy to the subcutaneous application of SPf66 malaria vaccine and placebo are reported for each dose of application in the participants of the vaccine efficacy trial in Brazil. Side effects evaluated two hours after each application were detected in 8.0%, 30.2% and 8.8%, for the Is', and 3"' dose, respectively, in the SPf66group, and in 7.0%, 8.5% and 2.9% in the placebo group. Local reactions such as mild inflammation, nodule and pain or erythema frequently accompanied by pruritus were the most common reactions detected in both groups (3-8%, 29.1% and 8.5% in the SPf66 group and 4.0%, 7.6% and 2.5% in the placebo group). Among vaccinees, local side effects after the 2nd dose were more frequent in females. Systemic side effects were expressed mainly through general symptoms referred by the participants and were most frequent after the 1st dose in both groups (4.3% in the SPf66 group and 3-0% in the placebo group). Muscle aches and fever were refewred by few participants. No severe adverse reactions were detected for either dose of application or group.

Effect of a booster-dose of rabies vaccine on the duration of virus neutralizing antibody titers in bovines

Humoral immune response using inactivated rabies vaccine was studied in 35 nelore cross-bred bovines of western region of São Paulo state. Ninety days after vaccination, 13 (92.8%) animals presented titers 30.5IU/ml, through mouse neutralization test. After 180 days, 9 (64.3%) sera showed titers 30.5IU/ml, after 270 days, only one (7.1%) showed a titer of 0.51IU/ml, and after 360 days, all animals showed titers < 0.5IU/ml. Group of animals receiving booster dose 30 days after vaccination presented, two months after, all with titers > 0.5IU/ml. At 180 days, 17 (80.9%) sera presented titers > 0.5IU/ml; at 270 days, 15 (71.4%), with titers 30.5IU/ml and at 360 days, 4 (19.0%), with titers 30.5IU/ml. Booster-dose ensured high levels of neutralizing antibodies for at least three months, and 240 days after revaccination, 71.4% of animals were found with titers 30.5IU/ml.

Low frequency of side effects following an incidental 25 times concentrated dose of yellow fever vaccine

In August/1999, a group of 14 adults from the staff of a private hospital in Contagem -- Minas Gerais State, Brazil, received unintentionally a 25 times concentrated dose of the 17-DD yellow fever vaccine (Bio-Manguinhos), due to a mistake at the reconstitution step. All patients were clinically and laboratorially evaluated at days 5, 13 and 35 post vaccination. Frequency of side effects and clinical observations of this group of individuals were not different from the observed in recipients immunized with normal doses of the vaccine. At the second and third evaluation none of the subjects reported symptoms. None of the patients presented abnormalities at the physical examination at none of the time points and in all cases the blood examination was normal, except for a reduced number of platelets that was detected in one subject at the first and second evaluation and reverted to normal at third evaluation. At the first evaluation point, 8 subjects were serum negative and 6 serum positive for yellow fever at the plaque reduction neutralization test. In 5 subjects the observed titre was 10 times higher as the baseline of 2.36 Log10 mUI/ml. The samples collected at second and third evaluation (13th and 35th days) demonstrated that all subjects responded to the vaccination with the exception of one that did not present a positive result in any of the samples collected. This evaluation confirms the safety of the 17-DD yellow fever vaccine.

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin®)

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin®), produced by Biobrás (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution. C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30µg of each protein at 15-day intervals combined with 250µg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis. The ability of these proteins to induce immune response and protection was analyzed. No statistical difference was observed in the level of IFN-g induced by proteins in vaccinated groups in comparison with control groups. Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14% were demonstrated for each purified protein.