Antonio Eugenio Castro Cardoso de Almeida

Multiplex-PCR serotyping of Listeria monocytogenes isolated from human clinical specimens

The genus Listeria is composed of six species of which Listeria monocytogenes is considered the single pathogenic species that causes listeriosis in humans. Of the 13 serovars of L. monocytogenes, 1/2a, 1/2b and 4b are responsible for the majority of clinical cases. The aim of this work was to detect L. monocytogenes in the cerebrospinal fluid sample of premature newborns and to characterize this sample using biotyping, serotyping and molecular typing. The results indicated the presence of L. monocytogenesin the clinical sample studied. Moreover, the isolate was identified as the 4b serovar that was characterized by the presence of a unique 691 bp band after analysis using the Multiplex-PCR technique. The results of repeated Multiplex-PCR and sequencing have indicated that the L. monocytogenes isolate was an atypical 4b serovar, which is the first time this finding has been reported.

Septic arthritis due to Haemophilus influenzae serotype a in the post-vaccination era in Brazil

Haemophilus influenzae (Hi) is an important bacterial pathogen in children, causing a variety of respiratory infections and lifethreatening diseases such as meningitis, epiglottitis, pneumonia and septicaemia. Occasionally, Hi can also cause sporadic infections such as septic arthritis (SA). Before the advent of Hi type b (Hib) vaccination, paediatric invasive Hi disease was caused mostly by Hib isolates. Hib SA in children had been responsible for a significant proportion of cases in Europe and the United States (Bowerman et al., 1997; Peltola et al., 1998; Shoaib et al., 2007).

Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples

BACKGROUND Several in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans. METHODS A total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae. RESULTS All used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains. CONCLUSIONS The in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.

Urinary tract infection caused by nontypable Haemophilus influenzae in the elderly.

Since the introduction of the Haemophilus influenzae serotype b (Hib) vaccine, the decrease in invasive Hib disease predominantly affecting children and the relative increase in nontypable H. influenzae (NTHi) infections have been well documented in many parts of the world. The increasing trend in the number of cases, especially among older adults, deserves attention (Heath et al., 2001; De Almeida et al., 2005; Dworkin et al., 2007; Tsang et al., 2007).

Characterization of strains of Neisseria meningitidis causing meningococcal meningitis in Mozambique, 2014: Implications for vaccination against meningococcal meningitis

Introduction In sub Saharan Africa, the epidemiology, including the distribution of serogroups of strains of N. meningitidis is poorly investigated in countries outside “the meningitis belt”. This study was conducted with the aim to determine the distribution of serogroups of strains of N. meningitidis causing meningococcal meningitis in children and adults in Mozambique. Methods A total of 106 PCR confirmed Neisseria meningitidis Cerebrospinal Fluid (CSF) samples or isolates were obtained from the biobank of acute bacterial meningitis (ABM) surveillance being implemented by the National Institute of Health, at three central hospitals in Mozambique, from January to December 2014. Serogroups of N. meningitidis were determined using conventional PCR, targeting siaD gene for Neisseria meningitidis. Outer Membrane Proteins (OMP) Genotyping was performed by amplifying porA gene in nine samples. Results Of the 106 PCR confirmed Neisseria meningitidis samples, the most frequent serotype was A (50.0%, 53/106), followed by W/Y (18.9%, 20/106), C (8.5%, 9/106), X (7.5%, 8/106) and B (0.9%, 1/106). We found non-groupable strains in a total of 15 (14.2%) samples. PorA genotypes from nine strains showed expected patterns with the exception of two serogroup C strains with P1.19,15,36 and P1.19–36,15 and one serogroup X with P1.19,15,36, variants frequently associated to serogroup B. Conclusion Our data shows that the number of cases of meningococcal meningitis routinely reported in central hospitals in Mozambique is significant and the most dominant serogroup is A. In conclusion, although serogroup A has almost been eliminated from the “meningitis belt”, this serogroup remains a major concern in countries outside the belt such as Mozambique.

Quality evaluation of commercially available male condoms in Rio de Janeiro, Brazil, 2009–2011

BackgroundThe increased incidence of sexually transmitted infections (STIs) in Brazil represents a significant public health issue. This issue has raised awareness among health authorities regarding the quality of condoms. In Brazil, male condoms need to be certified. The certification process evaluates in detail the manufacturing and quality of the final product; however, post-market surveillance is not part of the normal certification practice.MethodsFrom 2009 to 2011, we evaluated 20 male condoms brands per lot of 8 manufactures-both domestic and imported-marketed in Rio de Janeiro, Brazil. Sampling was performed per ISO 2859–1, and the condoms were evaluated on length, width, thickness, holes, integrity of primary packaging, bursting volume, bursting pressure, label and secondary packaging, following the criteria established in the Brazilian National Health Oversight Agency Resolution no. RDC 62/2008.ResultsOf the 20 evaluated brands, 17 brands were found to be noncompliant with the guidelines of the Brazilian National Health Oversight Agency Resolution no. RDC 62/2008 in at least one of the analyses performed.ConclusionsAny nonconforming unit has serious public health implications.

Changes in Haemophilus influenzae capsule locus: possible emergence of novel variants in Brazil.

A total of 28 strains of Haemophilus influenzae (Hi) a and b isolated from clinical samples before and after the introduction of the Hib conjugate vaccine in Brazil were analyzed to determine variants of the capsular gene. Our results suggest the occurrence of new variants closely related to types I and II previously described elsewhere. Eleven Hib strains belonged to type I, 8 were type II, and 3 Hia strains were type II. Six strains showed negative results after polymerase chain reaction targeting capsule locus; the variable regions were sequenced and compared with types I and II. Phylogenetic analysis showed that 5 Hib strains were actually subtypes of type I (type I-A), whereas 1 Hia strain was a subtype of type II (type II-A). Types I and II strains were present in both periods of vaccination. This study suggests that a gradual change in the capsule genes of H. influenzae is probably occurring, and novel variants might be emerging among Brazilian isolates.