Invasive non-type b haemophilus influenzae disease: report of eight cases / Doença invasiva causada por haemophilus influenzae não tipo b: relato de oito casos

ASTRACT Objective: To describe eight cases of invasive non-type b Haemophilus influenzae disease in children admitted to Hospital de Clínicas of Universidade Estadual de Campinas. Cases description: In 2015, there were eight cases of invasive non-type b H. influenzae disease. We tested the ampicillin sensitivity and beta-lactamase production of the strains identified and performed the genotyping. Molecular typing was determined by Pulsed-Field Gel Electrophoresis. Four patients were diagnosed with bacteremia; in two cases, H. influenzae was detected in the pleural fluid, and two patients had meningitis. Patients with comorbidities represented 37.5% of cases. Except for the strain of one patient - not sent to the reference laboratory -, all were ampicillin-sensitive and non-beta-lactamase-producing. Genotyping identified four non-capsular, one type c, and two type a strains. Molecular typing ruled out nosocomial transmission since all serotypes were distinct regarding genotype. Comments: The rise in cases of invasive non-type b H. influenzae infection was real. There was no nosocomial transmission, and we found no justification for the increase. These data indicate the need for surveillance to correctly diagnose, monitor, and understand the spectrum of non-type b H. influenzae disease.

ABSTRACT Objetivo: Descrever oito casos de doença invasiva por Haemophilus influenzae não tipo b em crianças internadas no Hospital de Clínicas da Universidade Estadual de Campinas. Descrição dos casos: Em 2015, ocorreram oito casos de doença invasiva por H. influenzae não tipo b. Nas cepas identificadas, testou-se a sensibilidade à ampicilina e a produção de betalactamase, e realizou-se a genotipagem. A tipagem molecular foi feita por Pulsed Field Gel Electrophoresis. Em quatro pacientes, o diagnóstico foi de bacteremia; em dois casos, H. influenzae foi identificado em líquido pleural, e dois pacientes tiveram meningite. Comorbidades foram encontradas em 37,5% dos pacientes. Com exceção da cepa de um dos pacientes (que não foi enviada ao laboratório de referência), todas eram sensíveis à ampicilina e não produtoras de betalactamase. A genotipagem identificou quatro cepas não capsulares, uma cepa tipo c e duas cepas tipo a. A tipagem molecular descartou a transmissão intra-hospitalar, já que todos os sorotipos eram distintos quanto ao genótipo. Comentários: O aumento dos casos de infecção invasiva por H. influenzae não tipo b foi real. Não houve transmissão intra-hospitalar e não foi encontrada justificativa para o aumento. Esses dados indicam a necessidade de vigilância para diagnosticar corretamente, monitorar e entender o espectro da doença causada por H. influenzae não tipo b.

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

Abstract 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.

Inflammatory response of Haemophilus influenzae biotype aegyptius causing Brazilian Purpuric Fever

The Brazilian Purpuric Fever (BPF) is a systemic disease with many clinical features of meningococcal sepsis and is usually preceded by purulent conjunctivitis. The illness is caused by Haemophilus influenza biogroup aegyptius, which was associated exclusively with conjunctivitis. In this work construction of the las gene, hypothetically responsible for this virulence, were fusioned with ermAM cassette in Neisseria meningitidis virulent strains and had its DNA transfer to non BPF H. influenzae strains. The effect of the las transfer was capable to increase the cytokines TNFα and IL10 expression in Hec-1B cells line infected with these transformed mutants (in eight log scale of folding change RNA expression). This is the first molecular study involving the las transfer to search an elucidation of the pathogenic factors by horizontal intergeneric transfer from meningococci to H. influenzae.

Multi locus sequence type comparison of invasive and commensal Haemophilus influenzae isolates from Delhi.

Haemophilus influenzae is a major public health concern in the developing world. The most virulent strain is H. influenzae Type b (Hib). Hib also constitutes a major portion of nasopharyngeal commensal flora in otherwise healthy individuals. Through dendogram based on composite gene sequences of seven multi locus sequence type genes, it was observed that invasive and commensal isolates made two completely separate clusters which are indicative of independent evolution of these two groups of H. influenzae in the Indian subcontinent.

Ribotyping, biotyping and capsular typing of Haemophilus influenzae strains isolated from patients in Campinas, southeast Brazil

Forty-five Haemophilus influenzae strains isolated from patients were characterized based on biochemical characteristics. Their capsular types were determined by polymerase chain reaction (PCR); they were compared, using two molecular methods [ribotyping with a specific DNA probe amplified from the 16S rDNA region from H. influenzae and through restriction fragment length polymorphism (RLFP) of an amplified 16S DNA region]. The strains were better discriminated by the ribotyping technique that used the 16S probe and by the combination of both techniques. Biotypes I and IV were the most common, followed by biotypes VI, VIII and III. Biotypes II and VII were not found. Most of the capsular samples were nontypable (89 percent), with capsular types a and b found in 2 and 9 percent of the samples, respectively. We concluded that there is a very close genetic identity among pathogenic and non-pathogenic strains.

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 percent, S. pneumoniae in 4 percent and H. influenzae in 1 percent of the 192 clinical samples assayed; 3 percent 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.

Effect of C-terminal 44 amino acids deletion on activity of Haemophilus influenzae UvrA protein.

UV-sensitive mutant strain of Haemophilus influenzae Rd MBH3, is 20 times more sensitive to UV irradiation than the wild type strain. The mutation responsible for increased UV sensitivity of the strain was identified as G --> A transition predicting synthesis of truncated UvrAdeltaC44 protein (Balsara & Joshi). Recombinant UvrAdeltaC44 protein was purified for the first time under denaturing conditions. The molecular weight of the recombinant protein was estimated as approximately100 kDa. Recombinant UvrAdeltaC44 protein was found to be less efficient in its ATPase and DNA binding activity as compared to the wild type protein. Recombinant plasmid carrying uvrAdeltaC44 gene could partially complement the UvrA deficiency in E. coli UvrA mutant.

Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae and Streptococcus sp. by polymerase chain reaction for the diagnosis of bacterial meningits

Avaliamos o desempenho da reação em cadeia da polimerase (PCR) para detecção simultânea da Neisseria meningitidis, Haemophilus influenzae e Streptococcus sp. no diagnóstico das meningites bacterianas e sua aplicabilidade na rotina diagnóstica. Foi realizado um estudo de coorte com 182 crianças apresentando suspeita de meningite bacteriana. Em 84, havia alterações clínicas e laboratoriais sugestivas de meningite bacteriana. Destas, 65 tiveram o agente etiológico identificado pelos métodos laboratoriais de rotina e 19 ficaram sem diagnóstico etiológico. Em 98 pacientes foi excluído o diagnóstico de meningite bacteriana. Analisando o desempenho da PCR encontramos sensibilidade de 88,1%, especificidade de 99,0% e valores preditivos positivo e negativo de 98,7% e 90,1% respectivamente. Nos 19 pacientes com meningite bacteriana mas sem diagnóstico etiológico a PCR detectou microrganismos em 14, sendo 12 N. meningitidis, um H. influenzae e um Streptococcus sp. A PCR possui o potencial de poder aumentar os índices de identificação das técnicas tradicionais, principalmente nas situações onde a microscopia direta, cultura ou identificação antigênica são negativos ou inconclusivos.

Tipificación capsular mediante PCR de aislamientos de Haemophilus influenzae no tipificables por aglutinación / PCR-based capsular typing of Haemophilus influenzae isolates non-typeable by agglutination

Haemophilus influenzae es reconocido como un agente patógeno responsable de infecciones localizadas y sistémicas. Se han descrito 6 tipos de polisacáridos capsulares antigénicamente distintos (a, b, c, d, e, y f ) que se pueden identificar por aglutinación en lámina con antisueros específicos. También existen cepas no capsuladas (NC) fenotípicamente no tipificables (NT). La introducción de la vacuna conjugada produjo una marcada disminución de las enfermedades invasivas causadas por H. influenzae tipo b. En este contexto, la tipificación capsular mediante PCR es el método más apropiado para distinguir las cepas no capsuladas de las mutantes b deficientes en cápsula (b-) y detectar la presencia de cepas pertenecientes a otros serotipos que no puedan ser tipificables por aglutinación. Se determinó el genotipo capsular a 38 aislamientos de Haemophilus influenzae no tipificables por aglutinación, derivados al servicio de Bacteriología Clínica del INEI-ANLIS "Dr. Carlos G. Malbrán" en el período 2002-2004. El 78,9% de los aislamientos provenían de hemocultivos y la mayor parte de ellos estaban asociados a foco respiratorio. El 100% de los aislamientos fueron identificados como H. influenzae no capsulados mediante la técnica de PCR.

Haemophilus influenzae is recognized as a pathogenic agent responsible of localized and systemic infections. Six antigenically different capsular polysaccharide types have been described (a, b, c, d, e, and f ) which can be identified by slide agglutination with specific antisera. Besides there are non capsulated strains that cannot be typed by slide agglutination. The introduction of the conjugated vaccine produced an important reduction of invasive diseases caused by H. influenzae type b. Capsular typing by PCR is the most appropriated method for distinguishing non capsulated strains from capsule deficient type b mutants (b-) and for detecting strains of other serotypes that cannot be detected by slide agglutination. Capsular genotype was studied in 38 isolates of non-typeable Haemophilus influenzae received at INEIANLIS "Dr. Carlos G. Malbrán" between 2002-2004. Of the isolates included in this study 78.9% of them were recovered from blood cultures and most of them were associated with a respiratory focus. By PCR technique 100% of the isolates were identified as non-capsulate H. influenzae and genotype b-was not detected.

An agent-based system for re-annotation of genomes

Genome annotation projects can produce incorrect results if they are based on obsolete data or inappropriate models. We have developed an automatic re-annotation system that uses agents to perform repetitive tasks and reports the results to the user. These tasks involve BLAST searches on biological databases (GenBank) and the use of detection tools (Genemark and Glimmer) to identify new open reading frames. Several agents execute these tools and combine their results to produce a list of open reading frames that is sent back to the user. Our goal was to reduce the manual work, executing most tasks automatically by computational tools. A prototype was implemented and validated using Mycoplasma pneumoniae and Haemophilus influenzae original annotated genomes. The results reported by the system identify most of new features present in the re-annotated versions of these genomes.

Detection Rates of Bacteria in Chronic Otitis Media with Effusion in Children

This study was performed to investigate polymerase chain reaction-based detection of bacterial DNA in middle ear fluid and assess the correlation between the PCR-positive rate with several factors associated with middle ear effusion. The purpose was to gain a further understanding of bacterial infection as a major cause of otitis media with effusion. Of the 278 specimens of middle ear fluid, 39 (14%) tested positive by ordinary culture. The overall detection rate of bacterial DNA using the PCR method was 36.7% for middle ear effusion, and bacterial DNA detection rates of Hemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis in the middle ear effusion were 29.1%, 4.7% and 10.8%, respectively. The bacterial DNA detection rate was higher in ears with a history of acute otitis media than those without the history. High detection rates were observed in patients younger than 48 months who have had a higher tendency to present with acute otitis media. We concluded that PCR is a more sensitive method for the detection of bacteria in middle ear effusion than ordinary culture, and acute otitis media is a major contributor to the pathogenesis of otitis media with effusion.

Ab initio gene identification: prokaryote genome annotation with GeneScan and GLIMMER.

We compare the annotation of three complete genomes using the ab initio methods of gene identification GeneScan and GLIMMER. The annotation given in GenBank, the standard against which these are compared, has been made using GeneMark. We find a number of novel genes which are predicted by both methods used here, as well as a number of genes that are predicted by GeneMark, but are not identified by either of the nonconsensus methods that we have used. The three organisms studied here are all prokaryotic species with fairly compact genomes. The Fourier measure forms the basis for an efficient non-consensus method for gene prediction, and the algorithm GeneScan exploits this measure. We have bench-marked this program as well as GLIMMER using 3 complete prokaryotic genomes. An effort has also been made to study the limitations of these techniques for complete genome analysis. GeneScan and GLIMMER are of comparable accuracy insofar as gene-identification is concerned, with sensitivities and specificities typically greater than 0.9. The number of false predictions (both positive and negative) is higher for GeneScan as compared to GLIMMER, but in a significant number of cases, similar results are provided by the two techniques. This suggests that there could be some as-yet unidentified additional genes in these three genomes, and also that some of the putative identifications made hitherto might require re-evaluation. All these cases are discussed in detail.

Comparative genomics using data mining tools.

We have analysed the genomes of representatives of three kingdoms of life, namely, archaea, eubacteria and eukaryota using data mining tools based on compositional analyses of the protein sequences. The representatives chosen in this analysis were Methanococcus jannaschii, Haemophilus influenzae and Saccharomyces cerevisiae. We have identified the common and different features between the three genomes in the protein evolution patterns. M. jannaschii has been seen to have a greater number of proteins with more charged amino acids whereas S. cerevisiae has been observed to have a greater number of hydrophilic proteins. Despite the differences in intrinsic compositional characteristics between the proteins from the different genomes we have also identified certain common characteristics. We have carried out exploratory Principal Component Analysis of the multivariate data on the proteins of each organism in an effort to classify the proteins into clusters. Interestingly, we found that most of the proteins in each organism cluster closely together, but there are a few 'outliers'. We focus on the outliers for the functional investigations, which may aid in revealing any unique features of the biology of the respective organisms

Gene HI1472 of Haemophilus influenzae Rd is a novel gene involved in DNA repair.

A chimeric plasmid, pJPuvr4, consists of a 16.7 kbp Haemophilus influenzae Rd chromosomal DNA insert at the EcoRI site of vector pJ1-8. This plasmid complements the UV and gamma ray sensitivity of the mutant strain MBH4. This plasmid carries the wild type allele of gene uvr4 which was localised to a 3.8 kbp DraI fragment, with an internal EcoRI site. Partial sequencing of the gene and its alignment with the published genome sequence of H. influenzae Rd revealed uvr4 to be HI1472. HI1472 is a putatively identified open reading frame (ORF), which has been assigned no function so far. The partial sequence did show nt database match with 3D exon of N cadherin gene of homosepians and moaA gene of H. influenzae. Cadherins are involved in cell adhesion, cell to cell contact and morphogenesis in homosepians and moaA gene codes for molybdenum biosynthesis subunitA. This report implicates HI1472 of Haemophilus influenzae Rd in DNA repair. Nucleotide sequence obtained for the gene uvr4 was compared with the published sequence of gene HI1472. A wild type strain variation was observed at the 592nd nucleotide position corresponding to a change from aspartic acid to threonine.

The minimal cellular genome of mycoplasma.

The mycoplasmas are the smallest and simplest self-replicating organisms, being built of a plasma membrane, ribosomes, and a circular double-stranded DNA molecule-the typical prokaryotic genome. The idea of using mycoplasmas as models for defining in molecular terms the entire machinery of a living cell was raised by Morowitz in 1984. The goal has been to prove the dogma of the completeness of molecular biology, that is, that the logic of life is finite, relatively simple and subject to full exploration. The recent complete sequencing of the genome of the human pathogen Mycoplasma genitalium brings us much closer to achieving this goal. The M. genitalium genome is only 580 kb long and contains only 470 predicted coding sequences(genes), as compared with 1727 in Haemophilus influenzae and about 4000 in E. coli. Thus, M. genitalium is apparently the simplest organism capable of independent life with a minimal set of genes. The drastic economization in genetic information must be associated with the parasitic mode of life of the mycoplasmas. Mycoplasmas evolved by reductive evolution from Gram-positive bacteria with low G + C genomes. During evolution the mycoplasmas have lost the cell wall and many biosynthetic systems involved in synthesis of macromolecule building blocks provided by their host. Thus, the M. genitalium genome carries only one gene involved in amino acid biosynthesis, and very few genes for vitamin and nucleic acid precursors; the lack of genes involved in fatty acid biosynthesis, leads to dependence on exogenous fatty acids, enabling the introduction of controlled variations in membrane acyl chains and the use of mycoplasmas as models in studying membrane fluidity. Moreover, the dependence of mycoplasmas on exogenous cholesterol for growth was exploited to show the role of cholesterol as a buffer of membrane fluidity. The mycoplasma genome carries the minimal set of energy metabolism genes, being content with a restricted supply of ATP needed for their parasitic mode of life. Being limited by a single permeability barrier enabled the saving of a considerable number of transport system genes. Nevertheless, these minimal organisms were shown to carry all the essential genes needed for DNA replication, transcription and translation, but even here gene saving is expressed in a minimal number of rRNA and tRNA genes. A genomic price had been paid to maintain parasitism, so that a significant number of mycoplasmal genes is devoted to adhesins, attachment organelles and variable membrane surface antigens directed towards evasion of the host immune system.

Chimeric plasmid transformation in Haemophilus influenzae.

Fate of 32P-labelled pJ1-8N19 DNA was followed in the mutant strain N19 and wild type H. influenzae Rd, during post-uptake incubation. Integration of the insert fragment carrying novr gene into the host genome was measured at various time intervals during post-uptake incubation. Negligible amount of label transfer and no detectable transfer of biological activity (novr) was observed in mutant strain N19 compared to wild type strain Rd. These observations correlated poor extra chromosomal establishments of the donor plasmid in the mutant strain N19.