Invasive Haemophilus influenzae disease in the vaccine era in Rio de Janeiro, Brazil.

BACKGROUND: Haemophilus influenzae (Hi) serotype b (Hib) conjugate vaccine was incorporated into the infant immunisation schedule in Brazil in 1999, where Hib was one of the major etiologic sources of community-acquired bacterial meningitis. OBJECTIVES: The purpose of this study is to describe the molecular epidemiology of invasive Hi disease in Rio de Janeiro state, Brazil, before and after vaccine introduction. METHODS: Surveillance data from 1986 to 2014 were analysed. Hi isolates recovered from cerebrospinal fluid (CSF) or blood from 1993 to 2014 were serotyped by slide agglutination, genotyped by multilocus sequence typing (MLST), and the capsule type evaluation, differentiation of serologically non-typeable isolates, and characterisation of the capsule (cap) locus was done by polymerase chain reaction. Antimicrobial susceptibility testing was performed using E-test. FINDINGS: From 1986 to 1999 and from 2000 to 2014, 2580 and 197 (42% without serotype information) confirmed cases were reported, respectively. The case fatality rate was 17% and did not correlate with the strain. Hib and b- variant isolates belonged to ST-6, whereas serotype a isolates belonged to the ST-23 clonal complex. Serotype a appeared to emerge during the 2000s. Non-encapsulated isolates were non-clonal and distinct from the encapsulated isolates. Ampicillin-resistant isolates were either of serotype b or were non-encapsulated, and all of them were ß-lactamase-positive but amoxicillin-clavulanic acid susceptible. MAIN CONCLUSIONS: Although Hi meningitis became a relatively rare disease in Rio de Janeiro after the introduction of the Hib conjugate vaccine, the isolates recovered from patients have become more diverse. These results indicate the need to implement an enhanced surveillance system to continue monitoring the impact of the Hib conjugate vaccine.

Three outbreak-causing Neisseria meningitidis serogroup C clones, Brazil(1.).

During 2003-2012, 8 clusters of meningococcal disease were identified in Rio de Janeiro State, Brazil, all caused by serogroup C Neisseria meningitidis. The isolates were assigned to 3 clonal complexes (cc): cc11, cc32, and cc103. These hyperinvasive disease lineages were associated with endemic disease, outbreaks, and high case-fatality rates.

Capsular switching in invasive Neisseria meningitidis, Brazil(1).

During the 1990s, an epidemic of B:4 Neisseria meningitidis infections affected Brazil. Subsequent increase in C:4 disease suggested B → C capsular switching. This study identified B → C switches within the sequence type 32 complex. Substantial disease related to capsular switching emphasizes the need for surveillance of circulating meningococcal strains to optimize disease control.

Detection of resistance genes and susceptibility patterns in Bacteroides and Parabacteroides strains.

Susceptibility to five antimicrobials was determined for Bacteroides spp. (n = 52) and Parabacteroides distasonis (n = 8). All isolates were susceptible to metronidazole. The resistance rates to ampicillin, cefoxitin, tetracycline and clindamycin were 98%, 9.6%, 65.3% and 19.2% of the Bacteroides strains, respectively. The genes cepA, cfiA, cfxA, tetQ, ermF and nim were found in 69.2%, 17.3% 9.6%, 50%, 7.7% and 3.8% for these strains respectively. All P. distasonis strains were resistant to ampicilin. Cefoxitin, tetracycline and clindamycin resistance rates were 75%, 87.5% and 50%, respectively. The ermF and nim genes were absent and 37.5%, 12.5%, 12.5% and 87.5% of this strains possessed cepA, cfiA, cfxA and tetQ genes, respectively. Ten cfiA gene positive strains of Bacteroides and Parabacteroides were submitted to E-test with imipenem and amoxicillin-clavulanate. The resistance rate to imipenem was 4.1% and 8.3% to amoxicillin-clavulanate. This feature is for the first time described in Brazil.

Neisseria meningitidis disease-associated clones in Amazonas State, Brazil.

BACKGROUND: The aim of this study is to describe the molecular epidemiology of Neisseria meningitidis invasive disease before the introduction of serogroup C conjugate vaccine in Amazonas State in 2010. METHODS: Meningococcal disease reported cases were investigated in Amazonas State during the period 2000-2010. N. meningitidis isolates (n = 196) recovered from patients were genotyped by multilocus sequence typing (MLST) and sequencing of porB, porA, fetA, fHbp and penA. Antimicrobial susceptibility was determined using E-test. RESULTS: In the study period, 948 cases were reported; the incidence was 2.8 for the entire state and 4.8 per 100,000 in the capital of Manaus. Most meningococcal disease was caused by N. meningitidis belonging to ST-32 (72%; 141/196) or ST-103 (21%; 41/196) clonal complexes. Capsular switching (B→C) was suggested within clonal complex (cc) 32. There were 6 (3%; 6/196) strains with intermediate susceptibility to penicillin and a single strain was resistant to rifampicin. Since 2007, serogroup C strains belonging to the cc103 have predominated and case-fatality has increased. CONCLUSION: We demonstrate a high rate of meningococcal disease in Amazonas State, where, like other parts of Brazil, serogroup C replaced serogroup B during 2000s. These data serve as a baseline to measure impact of serogroup C conjugate vaccine introduction in 2010. This study emphasizes the need for enhanced surveillance to monitor changes in meningococcal disease trends following the introduction of meningococcal vaccines.

Neisseria meningitidis: a neglected cause of infectious haemorrhagic fever in the amazon rainforest.

Neisseria meningitidis has not been seen as a significant cause of infectious haemorrhagic fever in the Amazon inlands; most reported cases are from the city of Manaus, the capital of the State of Amazonas. This picture is sustained by the lack of reliable microbiology laboratories, the perception of the health care workers, and the difficult to reach medical assistance; thus the number of confirmed cases is even lower with no reference of the strains phenotype. We report here the investigation of a case of suspected meningococcemia and his close contacts in a rural community in the Coari Lake, up the Amazon River.

Subclinical infection of the genital tract with Neisseria meningitidis.

We report the isolation of Neisseria meningitidis, characterized as B:NT:P1.7, from a female patient's genital tract in an outpatient clinic for HIV care. The gynecology clinic, as part of the follow up, collects specimens from all patients with HIV infection for routine exams and for early laboratory detection of sexually transmitted diseases . A Gram-negative diplococcus was isolated from the cervix of a heterosexual patient with AIDS. Based on this and other reported cases, urogenital infection with N. meningitidis can no longer be considered uncommon. The rising incidence of N. meningitidis isolated from this and similar sites has significant medical and diagnostic implications.

Non-toxigenic pattern II and III Bacteroides fragilis strains: coexistence in the same host.

Diarrhoeic stool samples from 334 0-5-year-old children were analysed with respect to the incidence of Bacteroides fragilis as well as other enteropathogens. B. fragilis was recovered in 9.3% (31/334) of the samples, and 79 strains were examined for the presence of the bft gene or the BfPAI flanking region using polymerase chain reaction assays. No enterotoxigenic B. fragilis strains were detected. In 29% (9/31) of the samples the coexistence of both II and III non-toxigenic B. fragilis (NTBF) patterns could be seen. In 51.6% (16/31) of the samples there existed a pattern II NTBF only, and in 19.4% (6/31) only pattern III could be detected. Strains from the same patient representing different patterns were submitted to pulsed-field gel electrophoresis assays. Fingerprints obtained by this technique showed that there was strong heterogeneity among strains from different individuals. However, different patterns from the same individual shared 100% similarity.

ß-Lactam resistance, serotype distribution, and genotypes of meningitis-causing Streptococcus pneumoniae, Rio de Janeiro, Brazil.

BACKGROUND: Here, we report a laboratory-based study of Streptococcus pneumoniae recovered from patients with meningitis in Rio de Janeiro State, Brazil. METHODS: The aim of this study was to determine the evolution of ß-lactam resistance, antimicrobial susceptibility pattern, serotypes, and genetic diversity of S. pneumoniae, isolated from meningitis patients between 2000 and 2008. RESULTS: A total of 264 S. pneumoniae recovered from patients between 2000 and 2008 were included. Susceptibility testing (E-test) of S. pneumoniae showed resistance to penicillin, ceftriaxone, oxacillin, cotrimoxazole, tetracycline, ofloxacin, erythromycin, chloramphenicol, and rifampicin. Penicillin resistance (PEN-R, minimal inhibitory concentration [MIC] ≥ 0.12 µg/mL) increased from 8% of isolates in 2000-2002, to 12% in 2003-2005, and to 20% in 2006-2008. Ceftriaxone resistance (MIC ≥ 1.0 µg/mL) was detected among some PEN-R isolates (13%) from 2004 onward. Within the PEN-R isolates, serotypes that are included in 10-valent pneumococcal conjugate vaccine predominated (90%), and resistance was detected mostly in isolates of serotypes 14 (61%), 23F (16%), 6B (10%), and 19F (3%). Multilocus sequence typing showed that 52% of the PEN-R isolates, and 89% of those with MICs ≥ 0.5 µg/mL, were sequence type (ST)-156 or single-locus variants of this ST (ST-557 or ST-4388); all of these isolates were serotype 14 and were assigned to the Spain-3 clone. CONCLUSIONS: ß-lactam resistance increased recently among cerebrospinal fluid isolates and was mainly due to the surge of the ST-4388, a previously undescribed gki single-locus variants of ST-156. Regional surveillance is shown to be essential to provide optimal antimicrobial therapy, monitor highly successful clones, and formulate adequate vaccination strategy.

Invasive Haemophilus influenzae disease in the vaccine era in Rio de Janeiro, Brazil

BACKGROUND Haemophilus influenzae (Hi) serotype b (Hib) conjugate vaccine was incorporated into the infant immunisation schedule in Brazil in 1999, where Hib was one of the major etiologic sources of community-acquired bacterial meningitis. OBJECTIVES The purpose of this study is to describe the molecular epidemiology of invasive Hi disease in Rio de Janeiro state, Brazil, before and after vaccine introduction. METHODS Surveillance data from 1986 to 2014 were analysed. Hi isolates recovered from cerebrospinal fluid (CSF) or blood from 1993 to 2014 were serotyped by slide agglutination, genotyped by multilocus sequence typing (MLST), and the capsule type evaluation, differentiation of serologically non-typeable isolates, and characterisation of the capsule (cap) locus was done by polymerase chain reaction. Antimicrobial susceptibility testing was performed using E-test. FINDINGS From 1986 to 1999 and from 2000 to 2014, 2580 and 197 (42% without serotype information) confirmed cases were reported, respectively. The case fatality rate was 17% and did not correlate with the strain. Hib and b- variant isolates belonged to ST-6, whereas serotype a isolates belonged to the ST-23 clonal complex. Serotype a appeared to emerge during the 2000s. Non-encapsulated isolates were non-clonal and distinct from the encapsulated isolates. Ampicillin-resistant isolates were either of serotype b or were non-encapsulated, and all of them were β-lactamase-positive but amoxicillin-clavulanic acid susceptible. MAIN CONCLUSIONS Although Hi meningitis became a relatively rare disease in Rio de Janeiro after the introduction of the Hib conjugate vaccine, the isolates recovered from patients have become more diverse. These results indicate the need to implement an enhanced surveillance system to continue monitoring the impact of the Hib conjugate vaccine.

The utility of the polymerase chain reaction assay for aetiologic definition of unspecified bacterial meningitis cases.

Most patients with acute suppurative meningitis are otherwise healthy individuals with regard to immune mechanisms against invasive bacterial disease. This medical emergency is among the most dramatic and potentially ravaging diseases that affect humans, particularly young children. The illness often strikes suddenly, and can either result in death or leave the survivors with significant neurological dysfunctions. The demonstration of a bacterial aetiology is necessary for decisions regarding treatment and prophylaxis. Conventional bacteriological methods frequently fail to identify an agent, as a result of administration of antibiotics or delayed lumbar punctures. We investigated the major aetiologic sources of unspecified bacterial meningitis cases (G00.9, ISCD-10) by polymerase chain reaction (PCR)-based identification of Neisseria meningitidis (crgA), Streptococcus pneumoniae (ply) and Haemophilus influenzae (bexA) in cerebrospinal fluid samples. The multiplex PCR detected N. meningitidis in 92%, S. pneumoniae in 4% and H. influenzae in 1% of the 192 clinical samples assayed; 3% were negative for all three DNA targets. Bacterial DNA detection was found to be a valuable adjunct to enhance bacterial meningitis surveillance when the yield of specimens by culture is reduced. The implementation of PCR assays as a diagnostic procedure in Public Health Laboratories is perceived to be a significant advance in the investigation of bacterial meningitis.

Recognition of the epidemiological significance of Neisseria meningitidis capsular serogroup W135 in the Rio de Janeiro region, Brazil.

Neisseria meningitidis retains its ability to cause endemic and hiperendemic disease in human population living in any environment, as well as localized outbreaks and massive epidemics in civilians and military personnel. In Rio de Janeiro it has been reported in the 1990s as prolonged outbreak of serogroup B and at least one epidemic of serogroup C was well defined, both demanding quick action by the Public Health authorities. We report here the emergence of serogroup W135 meningococcal disease causing endemic and case cluster in Rio de Janeiro during the first years of this new century.

Diagnosis of meningococcal meningitis in Brazil by use of PCR.

Fever and a petechial rash are strongly associated with meningococcal disease in the city of Rio de Janeiro. Early antibiotic therapy is indicated and, consequently, a reduction of confirmed cases by culture, Gram stain, and latex agglutination test is expected. We evaluated a multiplex PCR assay to identify Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae in biological samples from cases of non-culture proven meningitis with a petechial rash at presentation. To detect DNA in cerebrospinal fluid (n = 71) or blood (n = 5), a PCR screen was performed, based on the crgA, ply and bexA targets, respectively. Of the total, 70 CSF and 3 blood samples (96%) were positive by PCR for the presence of N. meningitidis DNA. Another PCR assay predicted in 82% of these samples N. meningitidis serogroups A (2%), B (60%), C (7%), X (3%), Y (2%), 29E (2%) or W135 (24%). In non-culture proven meningitis, PCR was found to be a valuable adjunct for the demonstration of meningococcal aetiology.

Assessment of a two-step nucleic acid amplification assay for detection of Neisseria meningitidis followed by capsular genogrouping.

Immediate prevention of meningococcal disease relies in part on the prompt treatment with antibiotics of household and other close contacts of cases; however intervention with effective vaccination relies on identification of serogroup-causing strains. Parenteral antibiotic for patient with suspected meningococcal disease before hospital admission is currently recommended. Laboratory standard methods are hindered by failure to detect bacteria by this medical approach to improve patient prognosis. We assessed two polymerase chain reaction (PCR) assays to detect (crgA) and define the serogroups (siaD, orf-2, and ctrA) of Neisseria meningitidis in 120 cerebrospinal fluid (CSF) samples from positive cases (culture or antigen detection or direct smear). The PCR sensitivity for the identification of N. meningitidis was 100% (95% confidence interval, CI, 96-100%) compared to a sensitivity of 46% for culture (95% CI 37-55%), 61% for latex agglutination test (95% CI 52-70%), and 68% for Gram stain (95% CI 59-76%); PCR specificity was 97% (95% CI 82-100%). PCR correctly identified the serogroups A, B, C, W135, Y, and X in CSF samples with a sensitivity of 88% (95% CI 80-93%); the primer sets were 100% specific. The introduction of PCR-based assays shall increase laboratory confirmed cases, consequently enhancing surveillance of meningococcal disease.

Diagnosis of Streptococcus pneumoniae meningitis by polymerase chain reaction amplification of the gene for pneumolysin.

Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96% (95% confidence interval, CI, 90-99%) compared to a sensitivity of 59% for culture (95% CI 49-69%), 66% for Gram stain (95% CI 56-74%), and 78% for latex agglutination test (95% CI 69-86%); PCR specificity was 100% (95% CI 83-100%). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.

The effect of subcapsular meningococcal B + C vaccine on the prognosis of patients with meningococcal disease.

The effectiveness of the meningococcal Cuban vaccine (VaMengoc B + C) was examined in terms of the prognosis of patients who develop disease. All cases in the vaccinee age category admitted to the Meningococcal Disease Reference Centre, Rio de Janeiro between August 1990 and December 1993 were enrolled. Vaccine effectiveness (VE) was estimated from the relationship 1-OR, where the OR (odds ratio) was the exponential of the logistic regression coefficient for the association between death from meningococcal disease and previous vaccination. The case fatality rate for vaccinees was 6.1% and that for non-vaccinees was 10.6% (relative risk 0.58; 95% confidence interval [CI] 0.33-1.01). An overall protective effect of the vaccine against a fatal outcome was identified (VE 53%; 95% CI 12-75%) controlling for sex, age at time of immunization, elapsed time since vaccination and time between onset of disease and hospital admission. This study suggests that, for some people, even if the vaccine does not protect against the development of disease it may have a beneficial effect in terms of preventing a fatal outcome. This protective effect needs to be further investigated in a prospective cohort study specifically designed to evaluate the new generation of meningococcal vaccines.