Lucimar G. Milagres

A multiplex PCR assay for simultaneous detection of Corynebacterium diphtheriae and differentiation between non-toxigenic and toxigenic isolates

The importance of identifying Corynebacterium diphtheriae can be appreciated since diphtheria remains endemic in many countries, alongside the risk of epidemic outbreaks and the existence of a large number of non-immunized people worldwide (Galazka & Robertson, 1995; Damasco et al., 2005). The features of infections caused by C. diphtheriae have changed over the decades, and are most clearly emphasized by the emergence of non-toxigenic strains causing atypical diseases, such as endocarditis, septic arthritis or infection in unusual anatomical sites (Mattos-Guaraldi & Formiga, 1998; Efstratiou & George, 1999; Reacher et al., 2000; Mattos-Guaraldi et al., 2003). Rapid and reliable methods are needed for identifying a C. diphtheriae infection, thus aiding in appropriate and timely patient management, and improved monitoring of cases.

Immunogenicity and safety of meningococcal C conjugate vaccine in children and adolescents infected and uninfected with HIV in Rio de Janeiro, Brazil.

Background: We aimed to evaluate the Meningococcal (Neisseria meningitidis) C conjugated (MCC) vaccine seroconversion and adverse events (AEs) in HIV-infected and HIV-uninfected children and adolescents in Rio de Janeiro, Brazil. Methods: HIV-infected or HIV-uninfected subjects, 2–18 years old, with CD4+ T-lymphocyte cell (CD4) percentage >15%, without active infection or antibiotic use, were enrolled. All patients were evaluated before and 1–2 months after immunization for seroconversion (defined as ≥4-fold titer increase in human serum bactericidal activity) and at 20 minutes, 3 and 7 days after immunization for AEs. Factors associated with seroconversion among HIV-infected group were studied. Results: Two hundred four subjects were enrolled: 154 HIV-infected and 50 HIV-uninfected. Median age was 12 years, and 53% were female. Among the HIV-infected group, 82 (53%) had a history of at least 1 C clinical category of Centers for Diseases Control and Prevention event, and 134 (87%) were using combination antiretroviral therapy. The median nadir CD4 percentage was 13% (0–47%). Seventy-six (37.3%) experienced mild AEs. Seroconversion occurred in 46 of 154 (30%) in the HIV-infected group and in 38 of 50 (76%) in the uninfected group (P < 0.01). Factors associated with seroconversion in the HIV-infected group were as follows: never had a C clinical category event [odds ratio (OR) = 2.1, 95% confidence interval (CI): 1.0–4.4]; undetectable viral load at immunization (OR: 2.4, 95% CI: 1.1–5.2) and higher CD4 nadir/100 cells (OR: 1.1, 95% CI: 1.0–1.2). Conclusion: MCC vaccine should be administered to HIV-infected children and adolescents after maximum immunologic and virologic benefits have been achieved with combination antiretroviral therapy. Our data suggest that a single dose of MCC vaccine is insufficient for HIV-infected individuals 2–18 years of age.

Antibody response to Pseudomonas aeruginosa in children with cystic fibrosis

Cystic fibrosis (CF) is the most frequent life threatening autosomal recessive disease in white subjects. The primary cause of morbidity and mortality in children with CF is chronic pulmonary infection, mainly caused by Pseudomonas aeruginosa. The purpose of this study was to assess the value of the measurement of antibodies to P. aeruginosa in diagnosing lung infection by the bacteria in CF patients. We assessed P. aeruginosa antibody titers in CF patients from Rio de Janeiro, Brazil, using cell lysate antigens as well as recombinant PcrV, a Type III Secretion System protein. Sputum (more than 70% of the specimens) or oropharyngeal swabs were obtained whenever patients were regularly followed for their pulmonary disease. Blood samples were obtained with an average interval of 6 months for a period of 2 years. The ELISA cut‐offs were assigned as the positive 95% confidence interval of the mean antibody levels from non‐fibrocystic controls. Our data showed that most CF patients (81%) of whom were not chronically infected by P. aeruginosa (Groups I and II), had their first serology positive for rPcrV. Cell‐lysate ELISA was able to detect P. aeruginosa antibodies before positive culture in the first serum sample of 44% of the patients from Groups I and II. When serum reactivity to rPcrV and cell lysate were combined, 94% of CF patients from Groups I and II (n = 16) had the first serology positive for P. aeruginosa over a mean time of 20 months before the first isolation of P. aeruginosa. In conclusion, longitudinal P. aeruginosa serology should become part of respiratory care follow‐up, in conjunction with other lung parameter functions. Pediatr Pulmonol. 2009; 44:392–401.

Comparison of PorA VR types and porA promoter sequence from Neisseria meningitidis B isolated from non-immunised children and vaccine failures immunised with a serogroup B outer membrane protein vaccine

PorA protein is an important component of group B meningococcal protein-based vaccines. The goals of this study were: (i) to classify the non-serosubtypable strains recovered from vaccine failures and controls by porA variable region (VR) type; (ii) to investigate if point mutations of VRs of the porA gene are present in P1.19,15 strains recovered from vaccine failures and controls; (iii) to investigate if nucleotide sequence variation in the promoter region of porA gene is related to low expression of PorA protein. VR type P1.19,15 predominated in younger vaccine failures (3-47 months) compared to older failures (48-83 months). No changes in VRs of porA were observed in 46 P1.19,15 strains studied. A promoter spacer of 16bp and 10 guanidine residues in the polymeric G tract was detected in five of six strains with weak PorA expression. Overall, this study indicated that lack of antibody response was probably the major cause of low vaccine efficacy in young children.

CD4+ T-cell activation impairs serogroup C Neisseria meningitis vaccine response in HIV-infected children.

Objective:To investigate the influence of CD4+ T-cell activation and regulatory populations in HIV-infected children antibody response to vaccination with a conjugate C polysaccharide vaccine. Design:CD4+ T-cell activation was evaluated by expression of CD38, HLA-DR and CCR5 molecules. Regulatory CD4+ T cells (TReg) were characterized as FoxP3+CD127−CD25+ and inducer T cells (TInd) as CD4+FoxP3−CD25−CD39+. Methods:All patients (n = 36) were HIV-vertically infected, aged 2–17 years-old and were vaccinated with one vaccine injection. Blood samples were obtained before and after immunization to determine bactericidal antibody titers (SBA), CD4+ T-cell activation and frequency of TReg and TInd subsets (multiparametric flow cytometry). Results:Children not-responding (n = 18) to MenC vaccine expressed higher frequency of activated CD4+ T cells (HLA-DR+CD38+CCR5+) than responders (n = 18), both before and after vaccination (P < 0.05). A significant higher frequency of TReg was detected in responders compared with nonresponders (P = 0.0001). We also detected an inverse correlation between CD4+DR+CD38+CCR5+ (P = 0.01) or CD4+DR+CD38+ (P = 0.02) T cells and TReg cell frequency after vaccination. CD4+ T-cell activation negatively correlated (P = 0.006) with postvaccination SBA titers but a positive correlation (P = 0.0001) was detected between TReg cells and SBA. TReg and TInd subsets were inversely correlated (P = 0.04). Conclusion:Our findings suggest that higher CD4+ T-cell activation leads to poor vaccine response in children living with HIV, which may be associated with a TReg/TInd disequilibrium.

Diphtheria-neutralizing antibody levels in healthy adults from Rio de Janeiro, Brazil

In Brazil, until 2004, the immunization policy against diphtheria involved childhood vaccination with no official routine booster dose administered after 15 years of age. This study assessed functional antibody levels against diphtheria among blood donors. A total of 140 blood samples were collected, and diphtheria antitoxin levels were evaluated by Vero cell neutralization test. The mean age of the population was 34 years old (range: 18-61 years); 37.8% females and 62.2% males. Overall, 30.7% (95%, CI: 23.4-38.7) individuals presented neutralizing antitoxin antibody titers < 0.01 IU/ml; 42.1% (95%, CI: 34.1-50.4) showed values between 0.01-0.09 IU/ml and, 27.1% (95%, CI: 20.2-34.9) had (3) 0.1 IU/ml. In the subgroup of individuals with history of diphtheria immunization during childhood (85%), a number of 28.5% showed unprotective levels of circulating neutralizing antibody (< 0.01 IU/ml). Despite the continuous progress of immunization programs directed to Brazilian population, currently healthy adults remain susceptible to diphtheria.

Human antibody and memory B and T-cell responses after primary and booster immunisation against Neisseria meningitidis B.

Vaccination against disease aims at the induction of long-lasting cellular and humoral immune responses. Few studies have addressed the mechanisms by which meningococcal vaccines generate and sustain immunological memory. The goal of this study was to investigate the development of long-term humoral and cellular memory to Neisseria meningitidis serogroup B (MenB) in health subjects after immunisation with the Cuban outer membrane protein (OMP) vaccine (VA-MENGOC-BC). The results showed that three doses of vaccine were necessary to induce a detectable memory B-cell response (mean of 0.46%) which became undetectable 6 months later. After boosting, only 2 of 5 individuals responded with an increase in memory B-cell frequencies (values of 0.15% and 0.34%). Bactericidal and opsonic antibody levels were higher after primary immunisation (log(2) mean and median of 4.7 and 1212, respectively) when compared with post-booster response (log(2) mean of 2.6 and median of 285, respectively). Together, these data suggest a failure of vaccine to induce long-term memory B-cell and serological memory in adults. However, we observed a significant and functional memory T-cell response specially after boosting, with a predominance of activated (CD69(+)) central memory T-cell (CD4(+)CD45(-)CCR7(+)) response. Therefore, this study suggests that vaccination with the MenB vaccine induced the generation and activation of memory T-cells but failed to maintain the memory B-cell population at a stable size and/or function.

Diphtheria toxin IgG levels in military and civilian blood donors in Rio de Janeiro, Brazil

Serologic data on diseases that are preventable by vaccines are necessary to evaluate the success of immunization programs and to identify susceptible subgroups. In the present study, we determined serum IgG levels against diphtheria toxin of military and civilian blood donors (N = 75; 69.3% males and 30.7% females) aged 18-64 years, from the Brazilian Army Biology Institute, Rio de Janeiro, using a commercial diphtheria kit (Diphtheria IgG ELISA; IBL, Germany). Most (63%) unprotected military donors were from the older age group of 41 to 64 years. In contrast, the majority (71%) of young military donors (18 to 30 years) were fully protected. About half of the military donors aged 31 to 40 years were protected against diphtheria. Among the civilians, about 50% of persons aged 18 to 30 years and 31 to 40 years had protective antibody levels against diphtheria as also did 64% of individuals aged 41 to 64 years. All civilians had a similar antibody response (geometric mean = 0.55 IU/mL) independent of age group. Military donors aged 18-30 years had higher IgG levels (geometric mean = 0.82 IU/mL) than military donors of 41-64 years (geometric mean = 0.51 IU/mL; P > 0.05). In conclusion, the existence of a considerable proportion of susceptible adults supports the position that reliable data on the immune status of the population should be maintained routinely and emphasizes the importance of adequate immunization during adulthood.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30 degrees C after growing for 16 h at 37 degrees C supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30 degrees C. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30 degrees C than from the cells after 16 h at 37 degrees C. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30 degrees C as from the cells after 16 h at 37 degrees C.

Comparison of long-term humoral memory development after immunisation against Neisseria meningitidis B or diphtheria toxoid.

Since genome sequence data became available there has been a marked increase in number of protein antigens that have been suggested as prospective vaccine components against Neisseria meningitidis B (MenB). Few studies have addressed the mechanisms by which meningococcal vaccines generate and sustain immunological memory. The goal of this study was to compare the B-cell response (antibody-secreting cells [ASC], memory B cell and IgG) evoked by a MenB vaccine (VA-MENGOC-BC(®)) with the B-cell response to diphtheria toxoid (DT) induced by a successful vaccine (Diphtheria-Tetanus-Pertussis [DTP]). The results showed different kinetics of specific ASC response after the primary and booster immunisations. Concerning the specific ASC kinetics, MenB vaccine induced a strong primary response, but the recall response showed a limited power over time. In contrast, DTP primary ASC response was weaker than the booster responses. We observed an increase in the relative percent of memory B cells after 1, 2 and 3 doses of MenB vaccine (mean of 0.8%, 1.3% and 1.6%, respectively) but without statistical significance. Similar frequencies were detected after boosting given at 4 months (mean of 1.3%) or 6 months (mean of 0.9%) following the third dose. DT specific memory B cell response showed a slight lower magnitude after the primary immunisation schedule (mean of 1.2% after the third dose) compared with the MenB response. However, a stronger memory B cell response was induced by booster doses of DTP vaccine at 4 months (mean of 1.9%) or 6 months (mean of 1.9%). The kinetics of specific IgG induced by both vaccines was similar, suggesting that memory B cells were responsible for the strong antibody response seen after the booster vaccination.